Although clinically approved hepatitis B virus (HBV) polymerase inhibitors (lamivudine‐3TC, entecavir, etc.) serve as effective therapeutics, the virus can easily generate resistance to them. Therefore, the treatment of HBV infection remains a public health problem. Numerous studies have shown that natural products have prospective anti‐HBV activity. The purpose of this study was to isolate and extract des(rhamnosyl) verbascoside from Lindernia ruellioides (Colsm.) Pennell and explore its anti‐HBV and hepatoprotective effects. Anti‐HBV activity was evaluated in HepG2.2.15 cells, a human hepatocellular carcinoma cell line with HBV‐stable infection, and its protective effect was evaluated in HL‐7702 cells, a normal human liver cell line. HepG2.2.15 cells maintained normal growth morphology within the selected concentration range of des(rhamnosyl) verbascoside. It also inhibited the expression of HBV antigens and HBV DNA in a dose‐ and time‐dependent manner in vitro. Further, western blot experiments showed that it could downregulate HBV X protein (HBx) expression in a dose‐dependent manner. In the H2O2‐induced hepatocyte injury model, the cell‐survival rate of the HL‐7702 cells with the highest drug dose reached 85.25%, which was significantly improved compared with that of the model group. Most of the cells returned to normal morphology, showing polygonal or fusiform structures. Thus, it may be stated that des(rhamnosyl) verbascoside exhibits anti‐HBV activity and hepatoprotective effects in vitro and may exert an anti‐HBV effect via antigen inhibition, HBV DNA secretion, and HBx protein expression.