Background: Deficiency of 25-hydroxyvitamin D (25OHD) is a stimulus for the secretion of parathyroid hormone (PTH). During surgery for primary hyperparathyroidism, 25OHD deficiency may artificially elevate PTH, decreasing the sensitivity of intraoperative PTH (ioPTH) measurements. Materials and Methods: Of 351 patients with known 25OHD status who underwent curative surgical treatment of primary hyperparathyroidism, 198 (56%) patients were 25OHD deficient (<25 ng/mL). A curative decrease in ioPTH was defined as a greater than 50% PTH decrease 5, 10, or 15 min after resection. Results: There was no statistical difference between 25OHD deficient and sufficient patients in PTH, phosphorous, and alkaline phosphatase preoperatively. There was a positive correlation between PTH and calcium, alkaline phosphatase, and gland weight, while there was an inverse correlation between preoperative PTH and 25OHD. The average ioPTH decrease was not significantly different after 5, 10, or 15 min, and 25OHD status did not affect when ioPTH indicated surgical cure. Conclusions: Lower 25OHD levels are correlated with higher PTH and alkaline phosphatase levels in patients with primary hyperparathyroidism. 25OHD status did not affect the average percent ioPTH decrease or the rate of cure. 25OHD deficiency does not affect ioPTH dynamics. © 2010 Society of Surgical Oncology.