Nuclear factor (NF)-kappaB is important for immune responses and cell survival; however, abnormal activation of NF-kappaB is linked with many types of diseases, including hepatocellular carcinoma (HCC). Our previous report indicated that hepatitis B virus (HBV) induces NF-kappaB activation through NF-kappaB-inducing kinase (NIK), and this can be blocked specifically by interferon (IFN)-gamma. In the present study, we report that HBV expression in HCC cell lines induces drug resistance against 5-fluorouracil (5-FU). This drug resistance was abolished by inhibition of NF-kappaB activation through small interfering RNA-mediated NIK 'knockdown' and IFN-gamma treatment. In addition to the reduced NF-kappaB activation and drug resistance, the upregulated growth arrest- and DNA damage-inducible protein 45beta (Gadd45beta) in HBV-expressing HCC cell lines was downregulated by the small interfering RNA-mediated NIK knockdown and IFN-gamma treatment. The overexpression of Gadd45beta in HCC cell lines also induces drug resistance against 5-FU. Based on our data, we suggest that IFN-gamma treatment might be helpful for chemotherapy in HBV-integrated HCC through inhibition of the NIK-mediated NF-kappaB activation and downregulation of the NF-kappaB target gene Gadd45beta.