The multistructural and multifunctional transmembrane glycoprotein CD44 is overexpressed in many tumors of distinct origin including malignant melanoma and contributes to a poor prognosis by affecting cell proliferation, cell migration, and also the sensitivity for apoptosis induction. Previous studies reported so far 15 CD44 regulatory microRNAs (miRs) in different cell systems. Using a novel method for miR affinity purification miR-143-3p was identified as most potent binder to the 3' untranslated region (UTR) of CD44. Overexpression of miR-143-3p in melanoma cells inhibits CD44 translation, which is accompanied by a reduced proliferation, migration and enhanced daunorubicin induced apoptosis of melanoma cells in vitro. Analyses of discordant CD44 and miR-143-3p expression levels in human melanocytic nevi and dermal melanoma samples demonstrated medium to high CD44 levels with no association to tumor grading or staging. The CD44 expression correlated to PD-L1, but not to MART-1 expression in malignant melanoma. Interestingly, the CD44 expression was inversely correlated to the infiltration of pro-inflammatory immune effector cells. In conclusion, the tumor suppressive miR-143-3p was identified as the most potent CD44 inhibitory miR, which affects growth characteristics of melanoma cells suggesting the implementation of miR-143-3p as as a potential anti-CD44 therapy of malignant melanoma.