The objective of this study was to explore the effect of mesenchymal stem cells (MSC) on HL-60 proliferation and its molecular mechanism. HL-60 cells co-cultivated with MSC were used as experiment group, and the cells cultivated solely were taken as control group. HL-60 cells in the two groups were counted at different time. The time-quantity curve was drawn. The cell cycle and apoptosis ratio of HL-60 cells were compared between the two groups and expressions of Survivin and Bcl-2 protein were detected by flow cytometry. The results showed that HL-60 cells cultivated with MSC were obviously inhibited, especially on day 5 and 7 (P < 0.01). HL-60 cells were distributed on the phase of G(0)/G(1) [control group (47.0 ± 9.0)% vs experiment group (70.0 ± 16.0)%, P = 0.003], and apoptotic peak appeared. Both of Survivin and Bcl-2 protein expressions in HL-60 cells decreased [Bcl-2 protein in control group (63.0 ± 9.1)% vs experiment group (50.0 ± 14.1)%, P = 0.045; Survivin in control group (94.0 ± 9.3)% vs experiment group (77.0 ± 11.8)%, P = 0.006]. It is concluded that the MSC can inhibit HL-60 cell proliferation, and promote HL-60 cell apoptosis.