Abstract: Objective To investigate the effect of precursor of nerve growth factor (proNGF) in promoting invasion of breast cancer cells and its relation with ezrin expression and phosphorylation of ezrin Thr567 and Tyr477. Methods Human breast cancer cell lines MDA-MB-231 and MCF-7 were stimulated by gradient concentrations of proNGF (0, 2.5, 5 and 10 ng/mL) for 16 h, and the invasion of the cells was assessed with Transwell assay. The expression of ezrin and the phosphorylation of ezrin Thr567 and ezrin Tyr477 in the treated cells were examined by Western blotting. MDA-MB-231 cells were transfected with pEnter-His-ezrinY477F (a dominant negative mutant) to study the role of phosphrylation of ezrin Tyr477 in the invasion of breast cancer cell stimulated by proNGF. Results proNGF significantly promoted MDA-MB-231 and MCF-7 cell invasion in a concentration-dependent manner (P<0.05), and concentration- and time-dependently increased the phosphorylation of ezrin Tyr477 (P<0.05) without affecting the expression of ezrin or the phosphorylation of ezrin Thr567. The specific inhibitor of src, SKI-606, significantly inhibited the phosphorylation of ezrin Tyr477 induced by proNGF. Transfection with pEnter-HisezrinY477F inhibited proNGF-induced invasion and phosphorylation of ezrin Tyr477 in MDA-MB-231 cells (P<0.05). Conclusion Phosphorylation of ezrin Tyr477 plays a critical role in the invasion of breast cancer cells stimulated by proNGF via proNGF/src/ezrin Tyr477 pathway.