Background: This study aimed to estimate stress markers, oxidative stress (OS), reproductive hormones and sperm parameters in male smokers and non-smokers and observe the impact of oxidative stress markers and smoking on sperm count, motility and morphology in a selected population of Karachi, Pakistan. Methods: This cross-sectional study was conducted from July 2017 to July 2018 at Aga Khan University (AKU), in Karachi, Pakistan. The subjects were recruited from the Sindh Institute of Reproductive Medicine (SIRM), Karachi based on defined inclusion criteria. The subjects were categorized into fertile and infertile based on cut off values of sperm parameters as recommended by the WHO i.e., sperm count/ejaculate of 39 × 106 /ml, sperm motility 40% and normal morphology 4%. Two hundred eleven fertile and 165 infertile male subjects were included in the study. Serum cortisol, adrenaline, superoxide dismutase (SOD), and glutathione peroxidase (GPX) were analyzed by ELISA kits. Data was analyzed on SPSS-22. A p-value of <0.05 was considered statistically significant. Results: Age, Body Mass Index (BMI), and body fat were similar among smokers and non-smokers. Age was significantly lower, while mean BMI and body fat were significantly higher among infertile smokers vs. fertile smokers (p-value < 0.05). The testosterone levels were significantly reduced among smokers as compared to non-smokers (p-value < 0.05). The median cortisol levels were increased as well as GPX, and steroid hormone-binding globulin (SHBG) were significantly reduced among smokers as compared to non-smokers. Additionally, the same findings with a significant difference have also been observed among infertile smokers as compared to fertile smokers (p-value < 0.05). This study has shown that the semen parameters (total count, motility, and morphology) are decreased in infertile smokers as compared to infertile non-smokers. Furthermore, the multivariate analysis showed that smoking causes a significant decrease in sperm count and morphology but it did not have any significant effect on motility. Conclusion: Smoking has a significant effect on fertility, specifically sperm count and normal morphology of sperm. This might be due to OS produced by smoking, which has.