Background Acanthosis nigricans (AN) is a prevalent dermatological issue with numerous treatment options that are not entirely satisfactory. Topical vitamin D analogues have been found to be effective and safe for treating AN; however, other preparations of vitamin D have not, to the best of our knowledge, been studied yet.Objectives To evaluate the safety and efficacy of intralesional vitamin D3 injection in treating pseudo-AN.Methods We enrolled 30 participants who had bilateral pseudo-AN of the neck. Every patient received intralesional vitamin D3 therapy on the right side of the neck and intralesional saline as a control on the left side over the course of four sessions, each spaced 1 week apart. Participants were assessed clinically 1 month after the last treatment session using the Acanthosis Nigricans Area and Severity Index (ANASI) score in addition to dermoscopic examination.Results A statistically significant difference in the ANASI score was seen between the treated and control sides after treatment, with the intralesional vitamin D-treated side exhibiting a larger percentage of reduction than the control side. Intralesional vitamin D3 treatment significantly improved dermoscopic features such as cristae cutis, sulci cutis and pigmented dots compared with the placebo treatment.Conclusions Intralesional vitamin D3 is a safe and effective modality for treating pseudo-AN. Acanthosis nigricans (AN) is a common problem with numerous treatment options that are not entirely satisfactory. Topical vitamin D analogues have been found to be effective and safe for treating AN; however, other preparations of vitamin D have not been studied yet. In this study, we evaluated the safety and efficacy of intralesional vitamin D3 injection for treating pseudo-AN in 30 participants. Every patient received intralesional vitamin D3 therapy on the right side of the neck and intralesional saline as a control on the left side over the course of four sessions, each spaced 1 week apart. A statistically significant difference in Acanthosis Nigricans Area and Severity Index score was seen between the treated and control sides after treatment.