This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5 × 10(4) cells/cm(2) in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5 × 10(5) cells/cm(2) could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz's L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The data and cells obtained from this study will contribute to development of in vitro model for basic biological studies using sturgeon species.