Background: Varicocele is one of the most common causes of infertility. Various modalities, such as retrograde blood flow accumulation of metabolites and the disturbance of thermoregulation in the testicles, mediate it. This study aimed to evaluate the effect of varicocele on hormonal cells (Sertoli and Leydig cells) by measuring the semen parameters and hormones before and after microsurgical varicocele ligation. Methods: A cross-sectional study was conducted among patients who underwent microsurgical subinguinal varicocele ligation from January 2023 to December 2023 at Gezira Hospital for Renal Disease and Urology (GHRDS). Any patient with abnormal semen analysis, grade-II or grade-III varicocele (palpable or visible) with reflux who underwent microscopic sub-inguinal varicocele ligation was included in this study. Results: The sample size was 54 patients.  The mean age of them was 36±7 years. Most of the patients had left-sided and grade-III varicocele (n=42; 78%). The degree of reflux was severe in most cases (n=25; 46%). The semen parameters including count, motility, and morphology were improved postoperatively (P=0.031). There was a significant increase in testosterone levels postoperatively (pre= 3.6±1.6 ng/ml V post= 5±1.9 ng/ml, mean difference= 1.4 ng/ml; P= 0.001) and decreasing in FSH levels postoperatively (pre= 17±15 mlu/ml V post= 13.6±12.1mlu/ml, mean difference= 3.4 mlu/ml; P= 0.000). Conclusion: Varicocele had an obvious effect on hormonal cells, confirmed by the good outcomes following microsurgical varicocele ligation, which led to an increased level of testosterone, reduction of FSH, and improvement of semen parameters.