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The present study aimed to evaluate the influence of serum vitamin D levels on semen quality and testosterone levels.This is a cross-sectional study conducted at Androscience,Science and Innovation Center in Andrology and High-Complex Clinical and Andrology Laboratory in Sao Paulo,Brazil,with 508 male patients,aged 18-60 years,from 2007 to 2017.Seminal parameters and serum sexual hormones were correlated with serum vitamin D concentrations in 260 men selected by strict selection criteria.Patients were divided into normozoospermic group(NZG,n = 124)and a group with seminal abnormalities(SAG,n = 136).Evaluation included complete physical examination,past medical history,habits and lifestyle factors,two complete seminal analysis with sperm functional tests,serum levels of 25-hydroxy-vitamin D3(25(OH)VD3),total and free testosterone,luteinizing hormone(LH),follicle-stimulating hormone(FSH),sex hormone-binding globulin(SHBG),total cholesterol,homeostatic model assessment of insulin resistance(HOMA-IR)index,and karyotype.The mean concentration of 25(OH)VD3 was significantly lower in the SAG(P＜0.001)and positively correlated with all baseline seminal parameters and total testosterone levels.In addition,serum vitamin D3 concentration was found to be positively correlated with sperm concentration(β= 2.103;P＜ 0.001),total number of spermatozoa with progressive motility(β= 2.069;P= 0.003),total number of motile spermatozoa(β= 2.571;P=0.015),and strict morphology(β = 0.056;P = 0.006),regardless of other variables.This is the first comparative study to address the issue of serum vitamin D3 content between normozoospermic patients and those with sperm abnormalities.It clearly demonstrates a direct and positive relationship between serum vitamin D level and overall semen quality,male reproductive potential,and testosterone levels.