Objective To investigate the effect of Sox9 gene on inducing the chondrogenic differentiation of human umbilical cord mesenchymal stem cells. Methods Human umbilical cord mesenchymal stem cells (hUCMSCs) were obtained by microdissection and digestion, and cultured in monolayer. The growth velocity and morphological changes of hUCMSCs were observed after Sox9 transfection. The expression of collagen Ⅱ and aggrecan were detected by immunohistochemistry. The mRNA expression of collagen Ⅰ, collagen Ⅱ and aggrecan were detected by RT-PCR, and collagen Ⅱ content was detected by western blot. Results The Sox9 gene had no significant effect on the morphology of hUCMSCs, and significantly accelerated the differentiation of hUCMSCs to chondrocytes compared with the traditional induction group. Conclusion Sox9 gene can regulate the differentiation of hUCMSCs, and hUCMSCs can be used as seed cells for cartilage tissue engineering.