The direct biological effects of radiofrequency electromagnetic radiation (RF-EMR) from wireless communication equipment on the testes are still unclear. Our previous study proved that long-term exposure to 2605 MHz RF-EMR gradually damage spermatogenesis and resulted in time-dependent reproductive toxicity by directly disrupting blood-testis barrier circulation. Although short-term exposure did not cause readily observable damage to fertility, whether it caused specific biological effects and how these effects contributed to the time-dependent reproductive toxicity of RF-EMR were currently unknown. Studies on this issue are important for elucidating the time-dependent reproductive toxicity of RF-EMR. The present study established a 2605 MHz RF-EMR (SAR=1.05 W/Kg) scrotal exposure model with rats and extracted primary Sertoli cells for exposure to investigate the direct biological effects of short-term RF-EMR exposure on the testis. The results showed that short-term RF-EMR exposure did not decrease sperm quality and spermatogenesis, but it increased the levels of testicular testosterone (T) and zinc transporter 9 (ZIP9) in Sertoli cells of rats. In vitro, 2605 MHz RF-EMR exposure did not increase the apoptosis rate of Sertoli cells, but it increased the apoptosis rate and MDA of Sertoli cells exposed to H2O2. T reversed these changes and increased ZIP9 level in Sertoli cells, whereas inhibiting ZIP9 expression significantly suppressed these T-mediated protective effects. Moreover, T increased the levels of phosphorylated inositol-requiring enzyme 1 (P-IRE1), phosphorylated protein kinase R (PKR)-like endoplasmic reticulum kinase (P-PERK), phosphorylated eukaryotic initiation factor 2a (P-eIF2a) and phosphorylated activating transcription factor 6 (P-ATF6) in Sertoli cells, and these effects were reversed by ZIP9 inhibition. With prolonged exposure time, testicular ZIP9 was gradually downregulated, and testicular MDA increased. ZIP9 level was negatively correlated with MDA level in the testes of exposed rats. Thus, although short-term exposure to 2605 MHz RF-EMR (SAR=1.05 W/kg) did not significantly disturb spermatogenesis, it suppressed the ability of Sertoli cells to resist external insults, which was rescued by enhancing the ZIP9-centered androgen pathway in the short term. Increasing the unfolded protein response might be an important downstream mechanism involved. These results promote a better understanding of the time-dependent reproductive toxicity of 2605 MHz RF-EMR.