Genetic causes of male infertility that is associated with aging are largely unknown. This study was designed to identify novel pathogenic variants of FOXP3 gene causing azoospermia. One homozygous (c.155 G > T) pathogenic variant of FOXP3 was identified in nine non-obstructive azoospermia patients, and one heterozygous (c.691 C > A) of FOXP3 was found in one non-obstructive azoospermia patient. Pedigrees studies indicated that the homozygous (c.155 G > T) FOXP3 pathogenic variant was inherited, while heterozygous (c.691 C > A) FOXP3 pathogenic variant was acquired. Human testis carrying pathogenic variant exhibited abnormal spermatogenesis. FOXP3 protein was expressed at a lower level or undetected in spermatocytes of mutant testis of non-obstructive azoospermia patients compared to obstructive azoospermia patients. FOXP3 stimulated the proliferation and inhibited the apoptosis of human spermatogonial stem cells, and we further analyzed the targets of FOXP3. We have identified two new pathogenic variants of FOXP3 in non-obstructive azoospermia patients with high incidence, and FOXP3 silencing inhibits the proliferation and enhances the apoptosis of human spermatogonial stem cells. This study provides new insights into the etiology of azoospermia and offers novel pathogenic variants for gene targeting of male infertility.