Previous studies indicated that NGX6, an EGFR negative regulating gene, can reverse the malignant phenotype of the colon cancer and down-regulate the expression of MADD(MAP-kinase activating death domain) which is an essential protein in the JNK pathway. All these results implied that whether NGX6 inhibits tumor growth by inactivating the EGFR-mediated JNK pathway? Western blot and immunohistochemistry were performed to detect the expression of EGFR, K-ras, p-JNK, c-Jun and cyclin D1 in the colon caner cell line and in the tissue of xenografts in nude mice, aiming at deciphering the effects of NGX6 on EGFR/ K-ras/JNK/c-Jun/cyclin D1 pathway both in vitro and in vivo. The results showed that NGX6 re-expression considerably suppressed the growth of xenografts in the nude mice. Data from Western blot revealed that NGX6 significantly down-regulated the expression of EGFR, K-ras, p-JNK, c-Jun and cyclin D1 in the colon cancer cells. And the further analysis by immunohistochemistry and Western blot in vivo indicated that NGX6 decreased the expression of EGFR, K-ras, p-JNK, c-Jun and cyclin D1 in the xenografts in nude mice，which were consistent with in vitro observations. Such evidences suggest that the major mechanism of NGX6 in colon cancer is negatively regulating EGFR-mediated JNK pathway, laying the experimental basis for further elucidating the mechanism of NGX6 gene.