The Red-eared Slider (Trachemys scripta elegans) is one of the world′s top 100 worst invasive alien species and has a strong ability to adapt to the environment. This study aims to establish a rapid and stable in vitro culture system of Red-eared Slider embryonic fibroblasts, and to provide material and method support for further study on the tolerance mechanism of Red-eared Slider to extreme environment at the cellular level. Red-eared Slider embryonic fibroblasts were isolated from stage 13﹣14 Red-eared Slider embryo by trypsin enzyme digesting and differential adherence, then were primarily cultured and subcultured in vitro. The growth curves of Red-eared Slider embryonic fibroblasts under different temperatures, different inoculation densities and different serum concentrations were drawn by MTT (Thiazolyl Blue Tetrazolium Bromide) method. The cultured Red-eared Slider embryonic fibroblasts were identified using reverse transcription-PCR (RT-PCR). Red-eared Slider embryonic fibroblasts were transfected using Lipofectamine 3000. The results showed that: 1) Red-eared Slider embryonic fibroblasts were typical spindle-shaped, irregular star or polygons. When reached to a certain density, they grew in multiple layers and arranged in a disorderly manner. With the increase of passage times, the cell volume increased and the spindle-like cells elongated. Red-eared Slider embryonic fibroblasts could be cultured for at least 15 generations (Fig. 1). 2) The growth state of Red-eared Slider embryonic fibroblasts was good at 30﹣34 ℃, and the growth rate increased with the increase of temperature. The inoculation density of Red-eared Slider embryonic fibroblasts should not be less than 1.25 × 104 cells/cm2 and the optimum serum concentration of the cell culture was 10% (Fig. 2). 3) The expression of vim and acta2, two fibroblast markers, were detected in the cultured Red-eared Slider embryonic fibroblasts by RT-PCR (Fig. 3). 4) Liposomes could successfully mediate the expression of exogenous green fluorescent protein and red fluorescent protein in Red-eared Slider embryonic fibroblasts, and the transfection efficiency was about 30% (Fig. 4). This study successfully isolated and cultured Red-eared Slider embryonic fibroblasts in vitro and the cells could be used for the transfection and expression of exogenous genes.