Objective To explore the role of the p21 gene in the migration and invasion of normal trophoblasts and the possible associated mechanisms. Methods The p21 gene in trophoblast HTR- 8 / SVneo was knocked down by siRNA. Cell proliferation, wound assays, and Transwell invasion assays were used to observe changes in the migration and invasion of trophoblasts with p21 knockdown. The effects of p21 knockdown on ERK3 and MMP2 expression were also examined at both mRNA and protein levels by real-time fluorescent quantitative PCR (qPCR) and western blotting. Results The migration and invasion of trophoblasts with p21 knockdown were significantly reduced, whereas there was no effect on cell proliferation. The expression of ERK3 and MMP2 was significantly decreased at both mRNA and protein levels. Conclusions These result indicate that the p21 gene can promote the motility of normal trophoblasts, and this effect is related to regulation of the expression of ERK3 and MMP2.