This study was designed to determine the effect of polyunsaturated fatty acids (PUFAs) present in flaxseed oil and soybean oil on fertility of male rats. Twenty-four male rats were divided into four groups each group contain six rats. The first group, the control group, received oil-free diet. The second group received diet contains flaxseed oil, the third group received diet contains soybean oil and the fourth group received diet contains both flaxseed oil and soybean oil with the ratio of 75% : 25%, respectively. Serum samples were collected for reproductive hormones analysis (LH, FSH and testosterone). Testicular tissues were collected with the head of epididymis for sperm cell count and semen quality test. Testicular tissues specimens were taken for histopathological examination, 17β-HSD3 gene expression and enzymatic activity as well as estimation of the antioxidant activity. The results of serum hormonal analysis showed a significant increase in testosterone and FSH levels without elevation in LH level. There was a significant increase in sperm cell count and semen quality in all three oil supplemented groups. Similar results were observed in gene expression analysis and enzyme activity of 17β-HSD3. Malondialdehyde (MDA) level was lower in all oil groups especially soybean oil group without significant differences between the four groups in nitric oxide (NO) level. Reduced glutathione (GSH) level was higher in oil groups than the control one. Glutathione S transferase (GST) activity was higher in group received both soybean and flaxseed oils without significant differences between the other groups including the control group. Finally, flaxseed and soybean oils have positive effects on spermatogenesis and steroidogenesis.