Enoyl-coenzyme A hydratase (ECHS1) is a key enzyme in the metabolism of fatty acids in mitochondria. We previously reported that hepatitis B surface antigen (HBsAg) interacted with ECHS1 in a yeast two-hybrid system. In the current study, we further examined their interaction by using GST pull-down and co-immunoprecipitation assays. The results confirmed that small hepatitis B surface antigen (SHBs) interacted with ECHS1. Furthermore, confocal imaging showed that SHBs and ECHS1 co-localized in HepG2 cells. To clarify the biological function of the interaction, human hepatoma cell lines that transiently and stably expressed SHBs were generated. The expression of SHBs led to a significant decrease in ECHS1 protein levels. ECHS1 protein levels were reduced to 48.44 ± 7.12 % in Huh7 cells transiently expressing SHBs, and to 54.97 ± 3.54 % in HepG2 cells stably expressing SHBs. In conclusion, our findings suggest that SHBs interacts with ECHS1 and regulates ECHS1 protein levels in hepatoma cells.