Background: Vitiligo is an acquired disease with unknown cause and one of the major challenges in dermatology. The aim of this study was to evaluate the therapeutic efficacy of microneedling in combination with topical liposomal form of 5-fluorouracil (5-FU) 5% in treatment of vitiligo.Methods: This study was performed as a randomized clinical trial on 25 patients with vitiligo referred to the dermatology clinics of Imam Reza and Ghaem hospitals in Mashhad City, Iran. In each patient, three lesions were selected and microneedling with liposomal form of 5-FU 5% and only microneedling were performed on one of them by random allocation. The third lesion was considered as a control without any intervention. This process was done every 2 weeks for 3 months, and the rate of improvement was assessed after 6 months.Findings: 68% of lesions (17 persons) treated with microneedling and 5-FU had significant repigmentation, 12% with 25%-50% improvement and 56% with improvement up to 25%. However, response rate of lesions treated with microneedling alone was 32%, 4% with 25%-50% improvement and 28% with improvement up to 25%. Statistical analysis of data with generalized estimating equation (GEE) method showed that in terms of response to treatment, the lesions treated with microneedling and 5-FU had a significant difference with microneedling alone and also with control lesions (P < 0.050).Conclusion: According to our findings, the combined use of microneedling with topical 5-FU significantly improves resistant vitiligo lesions compared to microneedling method alone.