We conducted this study to explore functions of TF II Aalpha/beta-like factor (ALF) during human spermatogenesis, and the relationship of its expression levels with male infertility. The RT-PCR and Western blot analyses illustrated that ALF was highly expressed in adult testis. Immunohistochemistry and immunoflurescence showed that ALF is located in the spermatid nuclei and in the annulus of spermatozoa. Further, to reveal whether ALF is related to male infertility, we performed the same experiments in infertility patients. The changes in the expression levels of ALF in the male infertility samples lead us to believe that ALF may function in spermatogenesis, especially in spermiogenesis. We also detected the ALF DNA methylation level by real-time methylation-specific PCR (MSP) both in testes of adult, fetal and infertile patient. The differential expression level of ALF gene in different types of testes was regulated by DNA methylation. Our research identified ALF as a human spermatogenesis related gene, the abnormal expression of ALF might be the partial cause for human infertility.