14-kDa phosphohistidine phosphatase (PHP14) was the first protein histidine phosphatase to be discovered, but its biological function remains unclear. In our previous study, we found that it was associated with tumor invasion. Here, we investigated its role in lung cancer cell migration and invasion. Knockdown of PHP14 expression in highly metastatic lung cancer CL1-5 cells inhibited migration and invasion in vitro, but did not alter cell proliferation rates. Overexpression of PHP14 in NCI H1299 cells promoted migration and invasion in vitro, but again did not alter cell proliferation. To evaluate the metastatic properties of PHP14 in vivo, an experimental metastasis assay was performed. Experimental metastasis in vivo was extensively inhibited by PHP14 knockdown. To further examine the mechanism underlying the involvement of PHP14 in cell migration, invasion, and metastasis, a comparative proteomics analysis was performed. The differential protein expression profiles revealed that PHP14 was probably involved in cytoskeletal reorganization; this was further supported by actin filament (F-actin) staining. These results demonstrate for the first time that PHP14 may be functionally important in lung cancer cell migration and the invasion of lung cancer cells, mediated partly through modulation of actin cytoskeleton rearrangement.