To investigate the regulation of endothelial cell (EC) microRNAs (miRNAs) altered by heat stress, miRNA microarrays and bioinformatics methods were used to determine changes in miRNA profiles and the pathophysiological characteristics of differentially expressed miRNAs. A total of 31 differentially expressed miRNAs were identified, including 20 downregulated and 11 upregulated miRNAs. Gene Ontology (GO) enrichment analysis revealed that the validated targets of the differentially expressed miRNAs were significantly enriched in gene transcription regulation. The pathways were also significantly enriched in the Kyoto Encyclopedia of Genes and Genomes analysis, and most were cancer-related, including the mitogen-activated protein kinase signaling pathway, pathways involved in cancer, the Wnt signaling pathway, the Hippo signaling pathway, proteoglycans involved in cancer and axon guidance. The miRNA-gene and miRNA‑GO network analyses revealed several hub miRNAs, genes and functions. Notably, miR‑3613-3p played a dominant role in both networks. MAP3K2, MGAT4A, TGFBR1, UBE2R2 and SMAD4 were most likely to be controlled by the altered miRNAs in the miRNA-gene network. The miRNA‑GO network analysis revealed significantly complicated associations between miRNAs and different functions, and that the significantly enriched functions targeted by the differentially expressed miRNAs were mostly involved in regulating gene transcription. The present study demonstrated that miRNAs are involved in the pathophysiology of heat-treated ECs. Understanding the functions of miRNAs may provide novel insights into the molecular mechanisms underlying the heat‑induced pathophysiology of ECs.