Gentamicin (GM) is an aminoglycoside that has harmful effects on the male germ cells and sperm quality. N-3 polyunsaturated fatty acids (n-3 PUFA) are natural antioxidants that influence cell signaling and inflammation. Heme-oxygenase-1 (HO-1) and heat shock proteins (HSP) aid in cellular protection against cellular insults. This study aimed to explore the potential alleviating influences of treatment with n-3 PUFA on GM-induced testicular damage. Thirty-two albino male rats were divided into four equal groups. (1) The control group received normal saline, (2) the n-3 PUFA group received 100 mg/kg body weight/day n-3 PUFA daily for 4 weeks, (3) the GM group received 100 mg/kg/day GM intraperitoneally for 10 consecutive days, and (4) the GM + n-3 PUFA group received intraperitoneal GM for ten days followed by treatment with n-3 PUFA for 4 weeks. Significant reductions in sperm motility, viability, serum testosterone, total testicular protein, and germinal epithelium height were observed in the GM-treated group, with upregulation of the oxidative stress markers, HO-1 mRNA, and HSP70, and downregulation of proliferating cell nuclear antigen (PCNA). We also observed cellular disorganization, vacuolation, tubular distortion, and a significantly higher percentage of collagen. Ultra-structurally, most of the spermatogenic cells were electron dense and degenerated with rarefied cytoplasm. Treatment with n-3 PUFA resulted in a significant increase in sperm motility, viability, serum testosterone, and in the germinal epithelium height. Upregulation of HO-1 mRNA, HSP70, and PCNA expression and a significant reduction in the oxidative stress index were also observed. The findings confirm the potential ameliorative role of and imply novel mechanisms by which n-3 PUFA protects against GM-induced testicular injury.