Serum vitamin D (VitD) levels have been inversely related with metabolic syndrome (MetS), although the direct impact of VitD is still debated. This study examined 879 subjects of working age from an obesity and occupational clinic in Milan, Italy. Among these participants, 316 had MetS, while 563 did not. A multiple logistic regression analysis was conducted to determine the odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) for MetS in relation to serum VitD levels. After controlling for age, sex, leisure time physical activity, and body mass index (BMI), individuals with VitD levels between 20 and 29.9 ng/dL, or at least 30 ng/dL, had approximately half the risk of developing MetS (OR: 0.52, 95% CI: 0.32-0.86 and OR: 0.50, 95% CI: 0.25-0.99, respectively) compared to those with VitD levels below 10 ng/dL. This study presents further evidence of the beneficial effect of adequate VitD levels on the risk of MetS in a population of overweight/obese workers, even after adjusting for BMI. This study supports the importance of testing for and-if required-supplementing VitD in individuals with metabolic risk factors.