Abstract Background This study will evaluate the effect of microneedling of vitiligo lesion with and without topical 5 fluorouracil. Vitiligo is an acquired pigmentary disorder of unknown etiology, affecting approximately 1 % of the world population, without predilection for race or sex. It is characterized by white macules and patches, whose size increases over time, due to the loss of melanocytes. Vitiligo can appear at any time, and it significantly impairs the patients’ quality-of-life Objective To compare the efficacy of microneedling followed by 5-FU with microneedling alone in the treatment of stable non-segmental vitiligo. Patients and Methods This interventional study was held in Dermatology, Andrology & STDs department, Faculty of Medicine, Ain Shams University during the period from January till November 2019. Fifty patients with stable non-segmental vitiligo were included in the study. Results Regarding the efficacy of treatment with microneedling only vs microneedling with 5-FU in our study, there is statistically significant difference between the two sides according to treatment response, needling with 5-FU side showed response in 76% of cases, while needling side alone showed (0%) response. The degree of improvement among our patients in microneedling with 5-FU side, we detect different grades of repigmentation as 12 patients of the studied participants (24%) showed no repigmentation, 23 patients (46%) showed mild grade (<25% repigmentation), 15 patients (30%) showed moderate grade (25–50% repigmentation) and no one of the studied participants experienced either good grade (50–75% repigmentation) either excellent grade (more than 75% repigmentation). Our study showed no relation between repigmentation response and both demographic and clinical data of the patients as (age, gender, family history, duration of disease, VASI severity score and Fitzpatrick skin phototype). On the other hand, we found statistically significant difference with better repigmentation after microneedling with 5FU on certain sites of vitiligo. We noticed that lesions on the trunk and extremities showed better results while the acral parts showed no response. Observing side effects after treatment, we noticed that, there is statistically significant difference in side effects incidence between two sides. Side treated with microneedling and 5FU experienced more frequent side effects as compared with the side treated with microneedling only, these side effects included, slight to moderate pain and burning sensation during procedure that was tolerated and disappeared within few minutes to hours and minimal erythema in the first few days post procedure. Conclusion On the basis of the current study, we can conclude that microneedling followed by topical 5% 5-FU of vitiligo lesion is safe and tolerable method of treatment of vitiligo. It can induce repigmentation in vitiligo lesion but it could not be considered as effective method of treatment. We need to combined it with other treatment modalities to improve the outcome. However, long followup of the patients is needed.