Notch signaling is involved in the early process of differentiation to determine the fate of stem cells. However, the precise role of Notch in human bone marrow‑derived mesenchymal stem cells (hBMSCs) remains unclear. The present study aimed to investigate the involvement of Notch signalling during the course of hBMSC differentiation into cardiomyocytes using hBMSCs, with multilineage differentiation ability, isolated and purified from human bone marrow. Flow cytometric analysis revealed that CD29, CD44 and CD90 were highly expressed on the surface of cells in their fifth passage, whereas detection of CD34, CD45, CD54 and HLA‑DR was negative. Visualization of morphological changes, western blotting, immunocytochemistry and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) demonstrated that hBMSCs differentiate into cardiomyocytes through treatment with 5‑azacytidine (5‑aza). Transmission electron microscopy revealed ultramicroscopic details of differentiated hBMSCs. Western blotting and immunocytochemistry demonstrated increased protein expression levels of α‑actin and cardiac troponin T expression, and RT‑qPCR revealed increased mRNA expression of Notch1 early in the process of differentiation (days 1, 4 and 7), and increased mRNA expression levels of the transcription factors GATA binding protein‑4 and NK2 homeobox 5 at day 28 day. In conclusion, differentiation of hBMSCs into cardiomyocytes was induced in vitro by 5‑aza, and was associated with upregulation of Notch1, GATA binding protein‑4 and Nkx2.5 expression. Overexpression of the Notch1 signaling pathway may represent a potential mechanism underlying the differentiation of hBMSCs.