Background: Male infertility is a global concern and it tends to increase due to miscellaneous factors, such as environmental toxins and genetic and lifestyle choices. The aryl hydrocarbon receptor (AHR) has recently attracted attention due to its involvement in male infertility mechanisms and impact on sperm production and function. AHR, a versatile receptor expressed in various tissues, including the testes, regulates the genes involved in spermatogenesis. AHR activation is associated with cell cycle regulation and chromatin condensation during spermatogenesis. Objectives: This study aimed to investigate the influence of AHR activation on blood-testis barrier (BTB) integrity, focusing on the role of tight junction protein-1 (TJP1) and exploring the effects of AHR modulation on spermatogenesis outcomes in adult male rats. Methods: Forty adult male rats were divided into four groups according to their treatment regimens. Briefly, the control group was kept without any treatment, the resveratrol (RES) group received an intraperitoneal (IP) injection of 100 mg/kg every 72 h and the AHR antagonist (AHR‾) group received an IP injection of CH223191 at a dose of 10 mg/kg every 72 h. Finally, the dimethyl sulfoxide (DMSO) group received DMSO which was used as a solvent for preparing RES and CH223191. The study lasted for 60 d to cover the entire spermatogenesis cycle. At the study’s endpoint, sperm chromatin maturity and condensation were evaluated in addition to Tjp1 gene expression in testicular tissue, supported by BTB integrity assessment. Results: The abnormal sperm chromatin maturity (ASCM) and condensation were significantly (P<0.05) higher in AHR‾ compared to all other groups. Tjp1 gene expression was significantly upregulated in the RES group compared to the control and AHR‾ group, which tightened the BTB and maintained the testicular homeostasis for normal spermatogenesis. Conclusion: RES treatment positively influenced sperm chromatin maturity, Tjp1 expression and BTB integrity, suggesting its potential as a protective agent against male reproductive health. Conversely, AHR antagonism leads to compromised sperm chromatin integrity and BTB function, highlighting the critical role of AHR in spermatogenesis and BTB maintenance. These results underscore the importance of AHR modulation in male fertility and provide insights into potential therapeutic interventions.