Background: Chemotherapeutic resistance in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC) patients is an unfortunate side effect of standard chemotherapy. This situation necessitates a better understanding of the molecular pathways underlying HBV + HCC chemoresistance in order to aid the development of novel chemotherapeutic targets. Methods: We generated two doxorubicin (DOX)-resistant HBV + HCC sublines HepG2.2.15 and Huh7 -1.3. qRT-PCR was used to evaluate dysregulation in hexosamine pathway genes in chemosensitive and chemoresistant HBV + HCC cell lines in vitro. Western blots, luciferase reporter assays, and in vivo xenograft tumor studies were conducted to reveal the role of the miRNA-325-3p/DPAGT1 axis in HBV + HCC chemoresistance. Results: The hexosamine pathway gene dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGTI) was found to be upregulated in both DOX-resistant cell lines. Enhancing DPAGTI activity significantly improved the survival of DOX-resistant cells. Silencing or pharmacological inhibition of DPAGTI inhibited xenograft tumor growth under DOX-treated conditions. DPAGTI upregulation was associated with higher levels of stemness-related markers and ATP-binding cassette (ABC) drug efflux transporters in DOX-resistant cell lines. miR-325-3p was found to negatively modulate DPAGTI expression and phenocopied the effects of DPAGTI silencing in vitro and in vivo. In HBV + HCC patients treated with transarterial chemoembolization (TACE), high and low levels of tumor DPAGTI and miR-325 -3p expression, respectively, were associated with a poor chemotherapeutic response. Conclusions: Our findings provide novel insights into the role of miR-325-3p/DPAGT1 axis dysregulation in supporting HBV + HCC chemoresistance. (C) 2019 Elsevier Inc. All rights reserved.