Metastasis is a major cause of mortality among lung cancer patients. In a previous high-throughput screening of 20,000 compounds to identify inhibitors of cancer cell migration, 4-methyl-3-nitrobenzoic acid was found to inhibit the migration of nine tumor cell lines. The present study used the human non-small cell lung cancer cell (NSCLC) treatment with 4-methyl-3-nitrobenzoic acid significantly inhibited epithelial growth factor (EGF)-induced chemotaxis and chemokinesis of cancer cells in NSCLC cells. The defect in chemotaxis is likely due to the impairment in EGF-induced cofilin phosphorylation and actin polymerization. Furthermore, EGF-induced cell adhesion, which is another key factor in cell migration, was also inhibited. Taken together, these results suggest that 4-methyl-3-nitrobenzoic acid is a potent inhibitor of cancer cell chemotaxis and may be developed into a novel antimetastasis drug.