Chronic infection with hepatitis B virus (HBV) is a major public health problem. Recently， RNA interfering-based strategy has shown great potential to eradicate HBV infection. In current study， we report the experimental observation of plant-derived artificial microRNAs (amiRNAs) acting as therapeutics in HBsAg transgenic mice. Two pieces of small silencing RNA sequences， siR471 and siR519， against HBV surface antigen gene (HBsAg) were designed and expressed in lettuce using plant endogenous microRNA biogenesis machinery. Administration of amiRNAs-containing lettuce decoction specifically inhibited the HBsAg gene expression. In long term treatments， the liver injury in HBsAg transgenic mice were alleviated and no toxicological effects were observed. Compared with synthetic siRNA， feeding amiRNAs at a lower level achieved a similar inhibitory effect on HBsAg expression in mice. These results strongly suggest that employing plant endogenous miRNA biogenesis machinery to generate medicinal siRNAs is a novel way to solve the problems of siRNA stability and reduce the potential side effects of RNAi therapy.