Aim To investigate whether all-trans retinoic acid(atRA) combined with collagen Ⅳ can promote mouse embryonic stem cells(mESC)to differentiate into smooth muscle cell. Methods The totipotency and chromosome of ES cells were examined by ALP staining, SSEA-1 staining, and in-vivo teratoma formation test. After forming EBs by suspending in EB media for 3 days, EBs were divided into four inducted groups:none induction(Control group); atRA group; collagen Ⅳ group; atRA+collagenⅣgroup. They were differentiated for 7 d and 14 d. Then, they were harvested at 7 d and 14 d to analysie by Western blot and immunostaining. Results ①The successful culture of the ESCs was identified by the totipotent function. ②Compared with the other three groups, the expression of SM-α actin in group atRA+collagenⅣ significantly increased(P<0.05). Meanwhile, the proportion of SM-α actin⁺/SM-MHC⁺ cells in group atRA+collagen Ⅳ was higher than the other three groups. Conclusion atRA combined with collagen Ⅳ could do better in inducing the differentiation of mouse embryonic stem cells to smooth muscle cell than both alone.