Objectives We evaluated the prevalence of 25-hydroxyvitamin D (25-(OH)D) deficiency in our setting according to season, sex, and age. We also studied the association with parathyroid hormone (PTH) levels. Methods The study population comprised all patients with requests for assessment of 25-(OH)D between January 1 and December 31, 2018, as registered in the database of the laboratory information system. Major exclusion criteria were pediatric samples (<18 years) and factors affecting 25-(OH)D and/or PTH levels (i.e., kidney injury, liver disease, PTH disorders). Results Among 33,601 patients (24,028 women, 9,573 men), the prevalence of 25-(OH)D deficiency was 48%. Prevalence was greater in males than in females (53% vs. 46%). By age group, deficiency was more prevalent in quartile 1 (Q1, 74–87 years) and less prevalent in quartile 2 (Q2, 60–73 years). By season, deficiency was greater in spring (nonsignificant differences with respect to winter) and lower in summer. The association between 25-(OH)D and PTH was assessed in 9,368 persons. Linear regression analysis showed a weak association (coefficient – 0.303). Multiple logistic regression analysis revealed a significant association between 25-(OH)D deficiency and increased PTH (Odds ratio (OR), 1.63). Other risk factors for increased PTH include female sex (OR, 1.27), season (winter, OR 1.63, spring OR 1.16), and age (quartile 1, OR, 3). Conclusions The prevalence of 25-(OH)D deficiency differed according to sex, age, and season of the year. Furthermore, elevation of PTH is mainly influenced by low 25-(OH)D, female sex, season, and age.