[Objectives] The Luopanshan Pig Sus scrofa domestica, an indigenous local breed of China, is found mainly in the Tongnan region of Chongqing. The population of purebred Luopanshan Pigs has significantly declined due to the widespread impact of African swine fever and intense competition from other breeds, putting them at risk of endangerment. To conserve this invaluable genetic resource, the establishment of a somatic cell genetic resource bank is essential. [Methods] In this study, we successfully established a fibroblast cell line from the ear margin of Luopanshan Pigs using the tissue block adhesion method. Fluorescent staining was utilized to detect mycoplasma contamination and to identify the fibroblast marker vimentin protein. Chromosomal karyotype analysis and G-banding were conducted to assess genetic stability. In addition, flow cytometry was employed to evaluate key cell characteristics, including apoptosis rate, cell cycle distribution, reactive oxygen species levels, and cellular senescence. Statistical analyses were performed using GraphPad Prism software (version 9.5), with one-way ANOVA, two-way ANOVA and Student’s t-test being used to determine significant differences. [Results] The study showed that primary fibroblasts migrated from tissue blocks within 2 to 5 d, exhibited an elongated, spindle-shaped morphology (Fig. 1). After 8 to 12 d of continued cultivation, the cell density reached 90%, allowing successful passaging. The first five generations exhibited robust growth, forming a dense monolayer within 2 to 3 d post-passage. However, from the sixth generation onwards, cell volume gradually increased, leading to an irregular morphology characterized by enhanced lamellipodia and filopodia formation, alongside a significant decrease in growth rate. Cell fusion was observed after approximately 4 to 5 d. (Fig. 2). To assess cell purity, fluorescent staining for vimentin (a fibroblast marker) and nuclear DNA was performed, revealing a fibroblast purity exceeding 98% in the third passages (Fig. 3). Hoechst staining confirmed the absence of mycoplasma DNA, indicating no contamination (Fig. 4). The Luopanshan Pig fibroblasts have a chromosome number of 2n = 38, comprising 18 pairs of autosomes. Specifically, there are five pairs of submetacentric chromosomes (chromosomes 1﹣5), two pairs of subtelocentric chromosomes (chromosomes 6 and 7), five pairs of metacentric chromosomes (chromosomes 8﹣12), and six pairs of telocentric chromosomes (chromosomes 13﹣18). In additional, there is one pair of sex chromosomes, which are classified as metacentric chromosomes (Fig. 5). The relative lengths of each chromosome are detailed in Table 2. The viability of the third-generation cells before cryopreservation and after recovery exceeded 85%, with no significant differences observed. The ninth generation showed a viability surpassing 80% before cryopreservation, the viability remained above 70% after recovery. Before cryopreservation and after recovery, the proliferation rate of third generation cells showed a rapid increase, following a standard “S” shaped growth curve. In contrast, the growth curve of the senescent cells of the ninth-generation cells showed a gradual increase in an “S” shape (Fig. 6). The results of the flow cytometry analysis showed a significant increase in the proportion of cells in the gap 1 phase (G1) phase after the third generation, compared to the conditions before cryopreservation, there was a slight decrease in the proportion of cells in the gap 2 phase (G2) and synthesis phase (S), but this difference was not statistically significant. Although there was a slight increase in the rate of apoptosis after recovery, this did not reach statistical significance. After the recovery of the ninth generation, there was a significant increase in the proportion of cells in G1 phases and a significant decrease in the percentage of cells in S phase. There was also a slight increase in the proportion of cells in G2 phase, but this difference did not reach statistical significance. Furthermore, a significant increase in the apoptosis rate was also observed (Fig. 7). Finally, compared to third generation cells, significantly increased levels of reactive oxygen species (ROS) and senescence were observed in ninth generation cells, with marked increases after recovery (Figs. 8, 9). [Conclusion] In this study, we successfully established a cell line of marginal fibroblasts from the ear tissue of Lopangshan pigs. By comparing the growth characteristics of third- and ninth-generation fibroblasts before cryopreservation and after recovery, we found that low-generation fibroblasts exhibited greater stability in terms of growth after recovery. Therefore, low-generation fibroblasts are more suitable for long-term ultra-low temperature preservation and provide stable and excellent materials for subsequent genetic studies compared to high generation fibroblasts.