Purpose: RUNX3 is a known tumor suppressor gene in several carcinomas. Aberration in RUNX3 expression has not been described for cutaneous melanoma. Therefore, we assessed the expression of RUNX3 in cutaneous melanoma and its regulatory mechanisms relative to tumor progression. Experimental Design: The expression of RUNX3 mRNA and miR-532-5p (microRNA) was assessed in melanoma lines and in primary and metastatic melanoma tumors. Results: RUNX3 mRNA expression was down-regulated in 11 of 11 (100%) metastatic melanoma lines relative to normal melanocytes ( P < 0.001). Among 123 primary and metastatic melanoma tumors and 12 normal skin samples, RUNX3 expression was significantly down-regulated in primary melanomas ( n = 82; P = 0.02) and in melanoma metastasis ( n = 41; P < 0.0001) versus normal skin ( n = 12). This suggested that RUNX3 down-regulation may play a role in the development and progression of melanoma. RUNX3 promoter region hypermethylation was assessed as a possible regulator of RUNX3 expression using methylation-specific PCR. Assessment of RUNX3 promoter region methylation showed that only 5 of 17 (29%) melanoma lines, 2 of 52 (4%) primary melanomas, and 5 of 30 (17%) metastatic melanomas had hypermethylation of the promoter region. A microRNA (miR-532-5p) was identified as a target of RUNX3 mRNA sequences. miR-532-5p expression was shown to be significantly up-regulated in melanoma lines and metastatic melanoma tumors relative to normal melanocytes and primary melanomas, respectively. To investigate the relation between RUNX3 and miR-532-5p, anti–miR-532-5p was transfected into melanoma lines. Inhibition of miR-532-5p up-regulated both RUNX3 mRNA and protein expression. Conclusions: RUNX3 is down-regulated during melanoma progression and miR-532-5p is a regulatory factor of RUNX3 expression.