Equine bone marrow–derived mesenchymal stem cells (BM-eMSCs) have been used in veterinary clinics and research worldwide. For clinical use, in vitro propagation of BM-eMSCs is required (at least 2 weeks) to yield sufficient cell number for transplantation. Many culture media have been used to propagate human and animal MSCs but there are very few comparative studies of equine mesenchymal stem cells (eMSCs). The best culture media must promote cell proliferation and maintain eMSC properties. The objective of this study was to compare five basic commercial culture media by examining the proliferation rate and a representative MSC gene expression profile. Five culture media Dulbecco's modified eagle medium (DMEM)-LG, DMEM-HG, minimum essential medium alpha (MEM α), RPMI-1640, and DMEM/F12 were compared. Cultured BM-eMSCs were collected at day 7 and day 14 to measure cell number and gene expression. Relative gene expression was performed using real-time RT-PCR. Our results showed that MEM α was significantly superior to other media (P < .05) in 14-day culture, enhancing the expression of eMSC genes (ITGB1, CD44 and POU5F1) but depressing differentiation genes (DCN, PPARG and ADIPOQ) in addition to promoting rapid cell proliferation when compared to the other media. We concluded that MEM α was the best media to propagate BM-eMSCs up to 14 days as it supported cell proliferation while maintaining eMSC gene expression. From this, we propose that MEM α may be the most suitable medium for short-term culture of BM-eMSCs for cell transplantation studies.