Leukemia is one of the most common malignancies in humans worldwide; however, the molecular mechanism of the effect of bone marrow mesenchymal stem cells (bMSCs) on leukemia cell growth remains unclear. The present study demonstrated that Wnt5a protein expression was significantly induced in bMSCs via an adenovirus vector (P<0.01). The results showed that the proliferation of HL60 cells, a leukemia cell line, was significantly inhibited when the cells were stimulated with the culture supernatant of adeno-Wnt5a bMSCs compared with the culture supernatants of bMSCs and adeno-vector bMSCs for 24 or 48 h (P<0.01). The promoted maturation levels of HL60 cells were also observed following stimulation with the culture supernatant of adeno-Wnt5a bMSCs (P<0.01). However, no significant difference was identified in the proliferation and maturation of HL60 cells among the three groups stimulated with the culture supernatants containing a neutralization antibody against Wnt5a. Furthermore, the bMSC-derived Wnt5a was found to influence the maturation and proliferation of the HL60 cells by enhancing the non-canonical Wnt signaling pathway, while inhibiting the canonical Wnt signaling pathway by upregulating the expression of receptor tyrosine kinase-like orphan receptor 2 and calcium/calmodulin-dependent protein kinase II, and suppressing the expression of β-catenin and cyclin D1. In conclusion, bMSC-derived Wnt5a modifies the proliferation and maturation of HL60 cells via activation of the non-canonical Wnt signaling pathway.