Recent studies on tissue-autonomous serotonin (5-hydroxytryptamine [5-HT]) function have identified new roles for 5-HT in peripheral organs. Most of these studies were performed by crossing mice carrying the Tph1tm1Kry allele with tissue specific Cre mice. In the present study, we found that 5-HT production was not completely abolished in Tph1tm1Kry KO mice. The residual 5-HT production in Tph1tm1Kry KO mice is attributed to the expression of a truncated form of TPH1 containing the catalytic domain. Hence, in an effort to obtain mice with a Tph1 null phenotype, we generated mice harboring a new Tph1 floxed allele, Tph1tm1c, targeting exons 5 and 6 which encode the catalytic domain of TPH1. By crossing the new Tph1 floxed mice with villin-Cre or insulin-Cre mice, we observed near-complete ablation of 5-HT production in the intestine and β cells. In conclusion, this improved Tph1 floxed mouse model will serve as useful and accurate tool for analyzing peripheral 5-HT system.