AbstractAll female vertebrates develop a pair of ovaries except for birds, in which only the left gonad develops into an ovary, whereas the right gonad regresses. Previous studies found that the transcription factor Paired-Like Homeodomain 2 (PITX2), a key mediator for left/right morphogenesis in vertebrates, was also implicated in asymmetric gonadal development in chickens. In this study, we systematically screened and validated the signaling pathways that could be targeted by Pitx2 to control unilateral gonad development. Integrated chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analyses indicated that Pitx2 directly binds to the promoters of genes encoding neurotransmitter receptors and leads to left-biased expression of both serotonin and dopamine receptors. Forcibly activating serotonin receptor 5-Hydroxytryptamine Receptor 1B (HTR1B) signaling could induce ovarian gene expression and cell proliferation to partially rescue the degeneration of the right gonad. In contrast, inhibiting serotonin signaling could block the development of the left gonad. These findings reveal a PITX2–HTR1B genetic pathway that guides the left-specific ovarian growth in chickens. We also provided new evidence showing neurotransmitters stimulate the growth of nonneuronal cells during the early development of reproductive organs well before innervation.