Abstract: Objective To observe the impact of miRNA6089 on the function of melanocytes and provide the genetic evidence of miRNA in regulating melanocytes to participate in the disease process of vitiligo. Methods In previous studies, miRNA microarray was utilized to screen for differential miRNAs between the plasma samples of vitiligo patients and healthy controls. Then, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was adopted to detect the differential expression of miRNA6089 in plasma from age-matched vitiligo patients and healthy controls. Primary melanocytes were extracted from human foreskin tissue, while miRNA6089 nucleic acid mimics (mimics) and inhibitors were chemically synthesized. Melanocytes were transfected with miRNA6089 mimics and inhibitors. The cells were divided into four groups: a Si-NC group (transfected with a control inhibitor), a Si group (transfected with a miRNA6089 inhibitor), a mimic-NC group (transfected with a control mimic), and a mimic group (transfected with a miRNA6089 mimic). Then, the proliferation of melanocytes was examined by cell counting kit-8 (CCK-8) assay. The differential expression of miRNA6089, murine double minute 2 (MDM2), solute carrier family 17 member 5 (SLC17A5), and Janus kinase 3 (JAK3) in each group was detected by RT-qPCR and Western blot. The potential targeting relationship between miRNA6089 and MDM2, SLC17A5, and JAK3 was evaluated using the HDOCK and PLIP platforms. Results Compared with the plasma samples of healthy controls, the expression of miRNA6089 in the plasma of vitiligo patients was significantly elevated. Compared with the Si-NC group, the Si group showed significant increases in proliferation activity, and the mRNA and protein expression of MDM2, SLC17A5, and JAK3. In contrast, compared with the mimic-NC group, the overexpressed mimic group presented significant decreases in proliferation activity, and the mRNA and protein expression of MDM2, SLC17A5, and JAK3. The binding affinity of miRNA6089 with MDM2, SLC17A5, and JAK3 was all favorable. Conclusions MiRNA6089 potentially exerts a negative regulatory role on the proliferation of melanocytes and melanogenesis, which is associated with mechanisms such as redox imbalance, abnormal melanin transport, and aberrant immune regulation.