To study the role of c-Jun N-terminal kinase (JNK) and its relation to transcription factor AP-1 and Jun family proteins in hepatocellular carcinoma (HCC) with or without hepatitis B virus (HBV) infection. Immunohistochemical and in situ hybridization techniques were performed for studying phosphorylated JNK (p-JNK), c-Jun, JunB, JunD and AP-1 in 40 cases of human HCC and corresponding nontumoral tissues. Positive staining of nucleus for p-JNK, c-Jun, JunD and AP-1 was presented in 28 (70%), 29 (72.5%), 32 (80%) and 25 (62.5%) in cancer cells respectively, while 0%, 28%, 17.5% and 10% in adjacent non-tumor tissues. The expression levels of p-JNK, c-Jun, JunD and AP-1 were significantly and positively correlated with each other and with HBsAg positive rate (P<0.05). JunB was negative staining in both cancer cells and non-tumor tissues of all cases. JNK phosphorylation may correlate with AP-1 activation and the expression of c-Jun and JunD in HCC. JNK/c-Jun/JunD/AP-1 signaling pathway may play an important role in the pathogenesis of HBV-associated HCC. JunB may not be involved in the process.