Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.