We have previously shown that mice exposed to chronic social defeat stress exhibit anxiety-like behavior and decreased serotonin transporter (SERT) expression and serotonin (5-HT) uptake in the prefrontal cortex (PFC). There is a correlation between these anxiety-like behavior and 5-HT uptake, suggesting that decreased SERT in the PFC may induce anxiety. In this study, to clarify whether the decrease of SERT in the PFC induces anxiety, we investigated the effect of SERT knockdown in the PFC on anxiety-like behavior. Retrograde adeno-associated virus (AAV) vectors producing shRNA against SERT were microinjected into the PFC, and after 10 days, the elevated plus maze test was performed to assess anxiety-like behavior. Mice treated with AAV producing shRNA against SERT showed decreased SERT expression and 5-HT uptake in the PFC, but no such changes in the nucleus accumbens. The elevated plus maze test revealed that mice treated with AAV producing shRNA against SERT exhibited anxiety-like behavior such as decreased time spent in the open arms. These results suggest that the decrease of SERT in PFC could induce anxiety.