Background Cold-inducible RNA-binding protein (CIRBP) is associated with cell stress. However, its upstream regulatory factors are still largely unknown. Objectives This study investigated whether CIRBP expression was regulated by transforming growth factor beta (TGF-beta) during the process of heat-induced testicular damage. Materials and Methods Ten male adult ICR mice were allocated to heat treatment (scrotal hyperthermia at 43 degrees C for 30 min, n = 5) and control group (n = 5); CIRBP and TGF-beta 1, TGF-beta 2, and TGF-beta 3 expression levels in the testis in mRNA and protein were analyzed. Then, we conducted in vivo and in vitro studies to investigate the regulatory effects of TGF-beta on CIRBP. In the in vivo study, male adult ICR mice were subjected to testicular hyperthermia followed by a local testicular injection of TGF-beta antagonist (non-selective TGF-beta I/II receptor inhibitor, 5 mu g or 10 mu g). In the in vitro study, GC2-spd cells were cultured under 43 degrees C for 30 min or with different TGF-beta isoforms (10 ng/mL), and CIRBP expression levels in the testis and GC2-spd cells were analyzed 24 and 48 h, respectively, after treatment. Results As a result, heat treatment significantly downregulated the relative CIRBP mRNA and protein expression (p = 0.006 and 0.011), and significantly upregulated TGF-beta 2 and TGF-beta 3 expression levels (p = 0.022 and 0.04, for mRNA, and p = 0.001 for both protein levels). Local testicular injection of 10 mu g TGF-beta antagonist significantly attenuated heat-induced histological damage to the testes and CIRBP downregulation (p = 0.038). Furthermore, TGF-beta 2 and TGF-beta 3 significantly downregulated CIRBP mRNA and protein expression in GC2-spd cells (all p < 0.01), exerting a similar effect to heat treatment. Discussion and Conclusion Our in vivo and in vitro experiments demonstrated that heat-induced CIRBP downregulation in the testes was mediated by the upregulation of TGF-beta. Further studies are needed to clarify the molecular mechanisms underlying these processes.