This study was aimed to investigate the protective effect of Wit3a gene modification on mouse bone marrow mesenchymal stem cells against the injury induced by Ara-C. The gene-modified MSC steadily expressing Wnt3a were established by adenovirus system. The acute direct damage effects of different concentrations of Ara-C on the unmodified MSC and the gene-modified MSC were assessed by using an in vitro culture system, and the corresponding controls were set. The proliferation and apoptosis of MSC exposed to Ara-C were detected by cell count kit-8 (CCK-8) and flow cytometry. The expression of BCL-2 protein related with cell apoptosis was assayed by Western blot. The results indicated that as compared with unmodified MSC, Ara-C exhibited a less inhibitory effect on the proliferation of gene-modified MSC. There was obvious difference between unmodified MSC and gene-modified MSC (p < 0.05). The proliferation of gene-modified MSC began to recover at 72 hours after removal of Ara-C. However, unmodified MSC showed sustained suppression of proliferation after withdrawal of Ara-C. In apoptosis, the apoptosis rate of gene-modified MSC induced by Ara-C was significantly lower than those of unmodified MSC (p < 0.05). In addition, the expression levels of BCL-2 protein in gene-modified MSC were up-regulated compared with unmodified MSC (p < 0.05). It is concluded that Wnt3a gene modification can significantly mitigate the damage of mouse bone marrow MSC induced by Ara-C.