Background Warts are common dermatological conditions caused by human papilloma virus. Most treatments for human papilloma virus rely on the destruction of involved tissue. The role of vitamin D injection in treating warts is thought to be via regulating cell proliferation and differentiation through the upregulation of vitamin D receptors and the induction of antimicrobial peptides. Objective To assess the clinical efficacy and safety of intralesional vitamin D in the treatment of cutaneous warts and also the significance of peripheral blood toll-like receptors 2 (TLR2) expression and serum levels of 25-hydroxyvitamin D in predicting the therapeutic success. Patients and methods In this prospective interventional study, 50 patients of cutaneous warts were subjected to history taking, proper dermatological examination, and peripheral venous blood collection for detection of TLR2 mRNA expression and a serum level of 25 (OH) D. Vitamin D3 was slowly injected into the base of the largest or the oldest wart at 2-week intervals. Results A total of 34 (69.4%) patients showed response to intralesional Vitamin D [21 (42.9%) showed complete resolution of all lesions, 13 (26.5%) showed partial clearing] and 15 (30.6%) patients showed poor or no response after four sessions. Serum level of vitamin D did not show a significant relation with treatment response. TLR2 expression was higher in the group showing complete response. Conclusion Intralesional vitamin D injection offers a safe and cost-effective therapy for different types of cutaneous warts. TLR2 expression in patients’ serum can be used to predict response to treatment by intralesional vitamin D, with better response in patients with higher expression.