Media with high concentrations of serum are commonly used to induce cardiomyogenic transdifferentiation in mesenchymal stem cells; however, serum contains numerous unknown growth factors and interferes with definition of specific cardiomyogenic transdifferentiation factors secreted from feeder cells. In the present study, we determined whether the transdifferentiation of human mesenchymal cells can be observed in a FBS-free medium. The efficiency of transdifferentiation was observed in 10% FBS-containing standard medium (10%FBS) and in FBS-free medium containing insulin and thyroxin (FBS-free). In the present study, we used human uterine endometrium-derived mesenchymal cells (EMC100, EMC214) and menstrual blood-derived mesenchymal cells (MMCs). After cardiomyogenic transdifferentiation, the efficiency and physiological properties of cardiomyogenesis (fractional shortening of the cell [%FS] and action potential [AP]) were evaluated. The efficiency of transdifferentiation in EMC100 and in MMCs increased 36%* and 163%* (*P < 0.05), respectively. The %FS in EMCs increased to 103%*. AP-duration more than 250 ms with a marked plateau was only observed in FBS-free (3/19), and not in 10% FBS (0/41). The cardiomyogenic transdifferentiation of human mesenchymal cells can be observed in the FBS-free medium. Phenotypes of generated cardiomyocytes were significantly more physiological in FBS-free than in 10% FBS.