Schwann cell precursors differentiating into a myelinating phenotype are critical for peripheral nerve development and regeneration. However, little is known about the underlying molecular mechanisms of Schwann cell differentiation. In the present study, we performed a cyclic adenosine monophosphate-induced Schwann cell differentiation model in vitro. Western blot analysis showed that p27(Kip1) expression was upregulated during the differentiation of Schwann cell, while the inhibition of p27(Kip1) expression by short hairpin RNA-mediated knockdown significantly abolished the expression of promyelinating markers and the alteration of cellular morphology. In addition, immunofluorescence revealed a decrease of p27(Kip1) nuclear staining and a concomitant increase of cytoplasmic staining in differentiated Schwann cells. In summary, our data indicated that p27(Kip1) was a positive regulator of Schwann cell differentiation in vitro.