Abstract The three FOXA transcription factors are mainly known for their roles in the liver. However, Foxa3-deficient mice become progressively sub/infertile due to germ cell loss. Because no data were available regarding the localization of the FOXA3 protein in the testis, immunohistochemistry was performed on mouse testis sections. In the fetal testis, a weak but consistent staining for FOXA3 is detected in the nucleus of Sertoli cells. In prepubertal and adult life, FOXA3 remains present in Sertoli cells of some but not all seminiferous tubules. FOXA3 is also detected in the nucleus of some peritubular cells. From postnatal day 20 onward, FOXA3 is strongly expressed in the nucleus of Leydig cells. To identify FOXA3 target genes in Leydig cells, MLTC-1 Leydig cells were transfected with a series of Leydig cell gene reporters in the presence of a FOXA3 expression vector. The platelet-derived growth factor receptor α (Pdgfra) promoter was significantly activated by FOXA3. The Pdgfra promoter contains three potential FOX elements and progressive 5′ deletions and site-directed mutagenesis revealed that the most proximal element at −78 bp was sufficient to confer FOXA3 responsiveness. FOXA3 from Leydig cells could bind to this element in vitro (electrophoretic mobility shift assay) and was recruited to the proximal Pdgfra promoter in vivo (chromatin immunoprecipitation). Finally, endogenous Pdgfra messenger RNA levels were reduced in FOXA3-deficient MLTC-1 Leydig cells. Taken together, our data identify FOXA3 as a marker of the Sertoli cell lineage and of the adult Leydig cell population, and as a regulator of Pdgfra transcription in Leydig cells. The FOXA3 transcription factor is differentially expressed in Sertoli and peritubular cells, as well as in the adult population of Leydig cells, and contributes to Pdgfra transcription in Leydig cells.