Melatonin is produced from tryptophan and is secreted by the pineal gland in periods of darkness; it has cytoprotective effects in endothelial cells. It has been reported that melatonin mediates the regulation of gene expression at the post-transcriptional level through its effect on miRNA expression. miRNAs are small non-coding RNAs that silence gene expression, regulate most cellular processes involved in development, and maintain cell functions. In this study, we confirmed the expression of 136 and 154 miRNAs when endothelial cells were treated with melatonin concentrations of 100 μM and 1 mM, respectively. We investigated anti-correlations in miRNA-target-mRNA pairs, and we identified a total of 13 and 44 mRNAs showing an expression difference of >1.5 fold, following exposure to melatonin concentrations of 100 μM and 1 mM, respectively. These profiling data revealed a variety of biological effects, including cell death, apoptosis and proliferation, via a Gene Ontology analysis of biological processes. The results of this study suggest that melatonin may regulate miRNA and gene expression and may have a cytoprotective effect on endothelial cells.