Background: Hypovitaminosis D and higher platelet numbers are emerging as cardiovascular risk factors, in particular in obese subjects. Methods: This observational study was aimed at investigating the relationship between platelet number and serum 25-hydroxyvitamin D (25(OH)D) levels in a cohort of individuals affected by overweight and obesity (body mass index (BMI) ≥ 25 Kg/m2). A sample of 341 subjects (248 women, 93 men), aged 18–71 years, taking no medication, was examined. Anthropometric, hormone, metabolic and common routine hematochemical parameters were examined and evaluated in association with platelet count and serum 25(OH)D levels. Results: Platelet numbers were inversely related to age (p < 0.04), 25(OH)D (p < 0.05) and uric acid (p < 0.04) levels, and directly associated with white blood cells (p < 0.01), Thyroid Stimulating Hormone (TSH) (p < 0.04), insulin levels (p < 0.002) and Homeostasis Model Assessment – Insulin Resistance (HOMA-IR) (p < 0.002). We applied statistical regression models to examine the relationship between platelet count (dependent variable) and parameters that had univariate associations with platelet numbers, showing that the association between platelet count and 25(OH)D was not confirmed. Moreover, vitamin D showed a negative independent association with BMI, diastolic blood pressure and serum insulin levels. Conclusions: This study indicates, for the first time, that vitamin D deficiency is associated with a parallel increase in platelet number, suggesting that higher platelet numbers may be one of the possible mechanisms leading to a greater cardiovascular risk in obese subjects. It also shows that vitamin D deficiency, a common condition in obesity, has independent associations with higher BMI, diastolic blood pressure and serum insulin levels.