Background: Serum concentrations of 25-hydroxyvitamin D [25(OH)D] are associated with the risk of several chronic and acute diseases. However, updated data on vitamin D status in Mediterranean countries, including Italy, remain limited, hindering effective public health strategies. Objective: To assess serum 25(OH)D levels and their seasonal variation in healthy blood donors aged 18–65 years living in Northern Italy and not taking vitamin D supplements. Given the latitude and high levels of environmental pollution, cutaneous vitamin D synthesis may be impaired in this population. Recent Italian guidelines on supplementation highlight the need for updated data on hypovitaminosis D prevalence and seasonal synthesis capacity. Methods: In this exploratory cross-sectional study, 534 blood donors (268 men and 266 women) attending the Transfusion Medicine Unit of Verona University Hospital were enrolled between April 2016 and May 2018. Serum 25(OH)D concentrations were analysed according to season. Clinical, lifestyle, pharmacological and dietary characteristics were also collected. Results: Among healthy, normal-weight individuals, the prevalence of vitamin D insufficiency (25(OH)D<50 nmol/L) was low and limited to one-two months per year. Overweight and obesity significantly reduced the likelihood of achieving adequate 25(OH)D levels through cutaneous synthesis for several months. Mean 25(OH)D concentrations were higher than those previously reported in the same area, while seasonal variation remained preserved. Conclusions: Despite persistent environmental pollution, seasonal vitamin D synthesis is not impaired in this Northern Italy population. Updated data show higher 25(OH)D levels compared to past studies, supporting current recommendations against routine supplementation in healthy normal-weight individuals under 70 years