Loss of E-cadherin expression in melanoma correlates with increased tumor thickness and reduced disease-free survival. The molecular mechanisms underpinning its differential expression in melanoma tissue remain elusive. MicroRNAs (miRNAs) have been implicated in tumor progression and regulation of E-cadherin expression. Here, we demonstrate a significant correlation between tumor thickness and loss of expression of miR-200a, miR-200c, and miR-203 in a series of 23 frozen primary melanomas, where it was confirmed in two subsequent validation series (series 1: six nevi, 15 primary melanomas, and 16 metastases; series 2: 11 matched pairs of primary melanomas and metastases). Decreased levels of miR-200a, miR-200c, and miR-203 correlated with increasing thickness in the combined validation series (P = 0.024, 0.033, and 0.031, respectively). In addition, progressive loss of miR-200a expression with disease progression was observed in series 1 (P < 0.001) and in series 2 (P = 0.029). MiR-200 in situ hybridization and E-cadherin immunohistochemistry demonstrated reduced expression of both at the deep invasive margin of the tumor. Furthermore, a functional validation study using an anti-miR200 strategy demonstrated that loss of miR-200 expression in melanoma cell lines reduced E-cadherin expression. Collectively, our data point towards an important role for miR-200 and miR203 expression in regulating E-cadherin during melanoma progression.