Background Literature regarding the effect of Roux-en-Y gastric bypass (RYGBP) on vitamin D level shows contradictory findings. Our goal was to determine preoperatively vitamin D levels, to evaluate the efficacy of therapeutic and prophylactic doses of vitamin D and to assess the relationship of 25-OH vitamin D level and body mass index (BMI). Methods We conducted a retrospective cross-sectional study of 72 patients who underwent RYGBP from Results Our study demonstrated that 80% of the obese patients undergoing RYGBP had serum 25-OH vitamin D levels of less than 32 ng/ml. Postoperative data show that 45% of these patients continue being vitamin D insufficient despite the treatment. We demonstrated that a statistically significant inverse correlation between BMI and 25-OH vitamin D levels (r=0.464, p=0.01) exists. Conclusion Our finding strongly supports the need for aggressive monitoring of vitamin D levels for long-term prevention of complications of vitamin D deficiency in gastric bypass patients. Identifying the factors that predict patient's responses to vitamin D supplementation requires larger-scale studies and further analysis of these tendencies suggested by our findings.