Apoptosis is an important regulatory event in testicular homeostasis and optimization of sperm production. Sertoli cells (SCs) form the blood–testis barrier creating a special microenvironment where germ cells develop and are under strict hormonal control. Estrogens and androgens are known to play critical roles in SCs functioning, improving their in vitro survival by preventing apoptotic progression. Herein, we studied the influence of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) on the apoptotic signaling pathways of immature rat cultured SCs. For that we chose key points of the apoptotic pathway that interact with the mitochondria and evaluated the mRNA expression and/or protein levels of several apoptotic markers such as p53, the anti-apoptotic protein Bcl2, the pro-apoptotic Bcl2 family member Bax, the apoptosis-inducing factor (AIF) and caspase-3 and 9. Caspase-3 activity and DNA fragmentation were also evaluated as endpoint markers of apoptosis. E2 and DHT down-regulated the mRNA transcript levels of p53, Bax, caspase-9 and caspase-3. The protein levels of AIF were reduced after DHT treatment while E2-treated cells presented decreased levels of cleaved caspase-9 protein. Moreover, Bax/Bcl2 ratio was significantly decreased in E2-treated cells. The apoptotic endpoints caspase-3 activity and DNA fragmentation presented significant decreased levels after hormonal treatment. Taken together, these results show that E2 and DHT act as apoptotic signaling modulators in in vitro immature rat SCs suggesting that androgens and estrogens may be capable of modulating independent pathways of the apoptotic event by regulating different pro-apoptotic factors.