Aim To improve the differentiation of bone marrow-derived mesenchymal stem cell (BMMSC) into cardiomyocyte-like cell; To observe the effect of intergrin-linked kinase (ILK) overexpression on the differentiation of BMMSC into cardiomyocyte like cell. Methods Swine BMMSC was isolated and transfected by recombinant adenoviral vectors containing both human wild-type ILK cDNA and humanized recombinant green fluorescent protein (GFP) (Ad-GFP-ILK). The differentiation of BMMSC into cardiomyocyte-like cell was induced with high concentration (50 mol/L) 5-azacytidine (5-AZA). Cells were divided into 4 groups:blank control group (N group), Ad-GFP transfection group (Ad group), 5-AZA inducing group (5-AZA group) and Ad-GFP-ILK transfection group (ILK group). Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell viability. Cardiac troponin I (cTnI) and α-actinin expressions were detected by immunocytochemistry after transfection for 2,4 weeks. Western blot was performed to assess the expressions of cTnI, gap junction protein connexin-43 (CX-43), caspase-3, and ILK after transfection for 4 weeks. Results MTT results showed that cell viabilities were significantly decreased in Ad group, 5-AZA group than those in N group, ILK group (P＜0.05), but there was no significant difference between Ad group and 5-AZA group, between N group and ILK group (P＞0.05). When the cells were cultured for 2 weeks, immunocytochemistry staining showed that 5-AZA group and ILK group began to express cardiac specific markers cTnI and α-actinin, while N group and Ad group had no obviously positive expression. Western blot results showed that the expression of ILK protein in ILK group was significantly higher than that in the other groups (P＜0.05). Compared with N group and Ad group, the expressions of cTnI in 5-AZA group and ILK group were significantly higher (P＜0.05), but there was no difference between 5-AZA group and ILK group, between N group and Ad group (P＞0.05). The expression trend of CX-43 in the 4 groups was the same as that of cTnI. Caspase-3 expressions in Ad group, 5-AZA group were significantly higher than those in other groups (P＜0.05). Conclusions ILK overexpression can promote the differentiation of BMMSC into cardiomyocyte-like cells, and the differentiation efficiency is not different from that of high concentration of 5-AZA stimulation in vitro. ILK overexpression has no effect on cell proliferation, and has a certain anti-apoptotic effect.