Thymosin β4 (TMSB4X) belongs to a class of highly conserved small proteins that are present in a high abundance in immune tissues, where it participates in various biological activities, including anti-inflammation, wound healing, apoptosis, and cell survival. However, little is known about the expression and regulation of TMSB4X in reproductive tissues. The aim of this study was to examine the expression of rat Tmsb4x and chicken TMSB4X genes during testicular and epididymal development. Rat Tmsb4x was strongly detected in the spermatogonia and spermatocytes of testis at early postnatal development and transmitted to Leydig cells at sexual maturation. Also, rat Tmsb4x was detected at an increased level in the epididymis during postnatal development. When compared to the rat, expression of the chicken TMSB4X gene was low in the testis and epididymal region, and the mRNA localization was also unexpected. Three experiments were performed to examine the regulation of rat Tmsb4x in the epididymis: after elimination of Leydig cells using ethylene dimethane sulfonate (EDS); after destruction of the testis by cryptorchidism; and after castration. EDS-treated and castrated rats were injected with testosterone propionate. The expression of Tmsb4x was significantly reduced in the epididymis of EDS-treated, and castrated rats. In contrast, Tmsb4x was significantly enhanced in the epididymis after testosterone treatment. The expression of rat Tmsb4x was also regulated in the epididymis after cryptorchidism. Collectively, the expression of Tmsb4x was strongly detected in the testis and epididymis of rats, and was highly regulated in the epididymis by testosterone. J. Exp. Zool. 319A: 584–595, 2013. © 2013 Wiley Periodicals, Inc.