miRNAs are key regulatory small non-coding RNAs involved in critical steps of melanoma tumorigenesis; however, the relationship between sequence specific variations at the 5' or 3' termini (isomiR) of a miRNA and cancer phenotype remains unclear. Deep-sequencing and qRT-PCR showed reduced expression of miR-144/451a cluster and most abundant isomiR (miR451a.1) in dysplastic nevi, in-situ and invasive melanomas compared to common nevi and normal skin (n = 101). miRNA in situ hybridization reproducibly confirmed lost miR-451a.1 in melanoma compared to nevus cells or adjacent keratinocytes. Significantly higher expression of miR-451a.1 was associated with amelanotic phenotype in melanomas (n = 47). In contrast, miR-451a was associated with melanotic phenotype, absent pagetoid scatter of intraepidermal melanocytes, superficial spreading histological subtype and tumor inflammation. Sequencing miRNAs from cultured melanocytes with cytoplasmic melanin gradient (light, medium to dark) showed absent miR-451a while revealing other melanin-associated miRNAs, e.g. miR-30b, miR-100 and miR-590 in darkly and let-7a, let-7i and let-7f in lightly to moderately pigmented cultured melanocytes. Ectopic expression of miR-144/451a in melanoma cell lines resulted in markedly higher levels of mature miR-451a.1 than miR451a or miR-144; and significantly retarded cell migration and inhibited invasion in a glucose-sensitive manner. Surprisingly, these effects were not mediated by calcium binding protein 39 (CAB39), a proven miR451a gene target. miR-144/miR-451a cluster is a novel miRNA locus with tumor suppressive activity in melanoma.