BackgroundInfertility impacts 16% of North American couples, with male factor infertility contributing to ∼30% of cases. Reproductive hormones, especially testosterone, are essential for spermatogenesis. An age-independent population-level decline in testosterone concentrations over the past few decades has been proposed to be a consequence of diet and lifestyle changes. Vitamin B12 is present in the testes and has been suggested as an adjuvant nutritional therapy for male infertility due to its potential to improve sperm parameters. However, evidence examining the relationship between vitamin B12 and reproductive hormones is limited. ObjectivesThe objective was to cross-sectionally examine the relationship between serum vitamin B12 and male reproductive hormones (luteinizing hormone, follicular stimulating hormone, total testosterone, estradiol, and prolactin). MethodsMen with infertility (n = 303) were recruited from Mount Sinai Hospital in Toronto, Canada. Serum was analyzed for vitamin B12 and reproductive hormones. Statistical analyses included nonparametric Spearman’s rank correlation coefficient, linear regression, logistic regression, and effect modification by age and BMI linear regressions. ResultsAn independent monotonic relationship between serum vitamin B12 and total testosterone (ρ = 0.19, P = 0.001) was observed. Serum vitamin B12 was linearly associated with total testosterone (unadjusted β = 0.0007, P = 0.008 and adjusted β = 0.0005, P = 0.03). Compared to individuals in the lowest tertile of serum vitamin B12, those in the middle tertile (adjusted odds ratio [OR] = 0.48; 95% confidence interval [CI]: 0.25, 0.93, P = 0.03) and the highest tertile (unadjusted OR = 0.41; 95% CI: 0.22, 0.77, P = 0.005 and adjusted OR = 0.44; 95% CI: 0.22, 0.87, P = 0.02) had reduced odds of testosterone deficiency. ConclusionsThese findings suggest that among men with infertility, low serum vitamin B12 is associated with a higher risk of testosterone deficiency and impaired androgenic hormonal profiles that impact spermatogenesis and consequently, fertility.