The choice of females to accept or reject male courtship is a critical decision for animal reproduction. Serotonin (5-hydroxytryptamine; 5-HT) has been found to regulate sexual behavior in many species, but it is unclear how 5-HT and its receptors function to regulate different aspects of sexual behavior. Here we used Drosophila melanogaster as the model animal to investigate how 5-HT and its receptors modulate female sexual receptivity. We found that knockout of tryptophan hydroxylase (Trh), which is involved in the biosynthesis of 5-HT, severely reduced virgin female receptivity without affecting post-mating behaviors. We identified a subset of sexually dimorphic Trh neurons that co-expressed fruitless (fru), in which the activity was correlated with sexual receptivity in females. We also found that 5-HT_1A and 5-HT₇ receptors regulate virgin female receptivity. Our findings demonstrate how 5-HT functions in sexually dimorphic neurons to promote virgin female receptivity through two of its receptors.