Metastasis is the major cause of morbidity and mortality from breast cancer. Cell motility and chemotaxis play important roles in the metastatic cascade of cancer cells. Protein kinase C ζ (PKCζ) mediates cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. In the current study, we investigated the inhibitory effect of a compound called J-4 targeting PKCζ. J-4 was tested with inhibitory concentration (IC(50)) of 10 µmol/l using a Z'-LYTE™ Kinase Assay-Ser/Thr 7 Peptide Kit. Our results show that J-4 inhibited spontaneous migration and epidermal growth factor (EGF)-induced chemotaxis of human breast cancer cell MDA-MB-231. Through an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the drug designated as J-4 had no obvious cytotoxicity in vitro. Meanwhile, in the presence of J-4, the cells showed defects in EGF-induced actin polymerization and adhesion. Furthermore, J-4 dampened EGF-induced phosphorylation and recycling of cofilin. Taken together, our data demonstrate that J-4 is a new and typical inhibitor that blocks the PKCζ pathway. Moreover, a better understanding of the mechanism of action of J-4 may provide a novel medical therapeutic strategy for cancer treatment that would block metastasis, thereby reducing the proliferation and dissemination of cancer cells and increasing patient survival.