Piperine is a major alkaloid of black pepper (Piper nigrum Linn.) and long pepper (P. longum Linn.), and its antidepressant-like effect has been previously demonstrated. The purpose of this study was to explore the possible contribution of the serotonergic system in the antidepressant-like effect of piperine in mice. The results showed that piperine significantly reduced the immobility time in the forced swim test and tail suspension test in mice. The anti-immobility effect of piperine in the forced swim test and tail suspension test was completely abolished by pre-treating the mice with pCPA (an inhibitor of 5-HT synthesis). Piperine treatment also significantly potentiated the number of head-twitches of mice induced by 5-HTP (a metabolic precursor to 5-HT). In addition, the neurochemical assays showed that piperine produced a marked increase of 5-HT level in both the hippocampus and frontal cortex of mice. Taken together, these results clearly suggest that the antidepressant-like effect of piperine is mediated via the serotonergic system by enhancing 5-HT content in mouse brain.