Background: Vitamin D plays a pivotal role in calcium and phosphorus metabolism, also influencing bone tissue. Several studies have reported that vitamin D blood levels were significantly lower in people with obesity, probably due to its uptake by the adipose tissue. Clinical studies that investigated the changes of circulating levels of vitamin D following weight loss reported controversial data. A very low-calorie ketogenic diet is acknowledged as a reliable treatment to achieve a rapid weight loss. Therefore, we investigated the effect of weight loss, consequent to a very low-calorie ketogenic diet, on vitamin D blood concentrations. Methods: A cohort of 31 people with obesity underwent a very low-calorie ketogenic diet for 10–12 weeks. The serum concentrations of vitamin D, parathormone, calcium and phosphorous were measured before and after weight loss; they were compared to a control group of 20 non-obese, non-diabetic, age- and gender-matched persons. Results: Patients with obesity had a higher habitual intake of vitamin D than the control group (p < 0.05). However, the vitamin D blood levels of the obese group were significantly lower than those of the control group (p < 0.005) and they increased after weight loss (p < 0.001). At baseline, vitamin D blood concentrations of the persons with obesity were significantly correlated with both fat mass–kg (r = −0.40; p < 0.05) and body mass index (r = −0.47; p < 0.01). Following very low-calorie ketogenic diet, the change in vitamin D serum concentrations was correlated only with the change in fat mass–kg (r = −0.43; p < 0.01). Conclusion: This study confirmed that patients with obesity have lower vitamin D levels that normalize after significant weight loss, supporting the hypothesis that vitamin D is stored in the adipose tissue and released following weight loss.