The present study aimed to investigate the microRNA (miRNA) profile in human medullary thyroid carcinoma (MTC) tissue. The GSE40807 data profile was downloaded from the Gene Expression Omnibus database. Following preprocessing, differentially expressed microRNAs (DEMs) between MTC and healthy tissues were identified. Based on the obtained DEMs, transcription factor (TF) -miRNA and miRNA-target gene regulatory association pairs were predicted. Finally, functional enrichment analysis was performed on target genes of DEMs. Fifteen upregulated and 17 downregulated DEMs were identified. In the constructed TF-miRNA regulatory network, hsa-miR-9-5p was regulated by 9 TFs and hsa-miR-1 was regulated by 8 TFs. TFs of nuclear factor of. light polypeptide gene enhancer in B-cells 1 (NF-kappa B1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) regulated 4 and 3 DEMs, respectively. In the miRNA-target gene regulatory network, hsa-miR-1, hsa-miR-9-5p, hsa-miR-96-5p and hsa-miR-590-5p were most upregulated. The target genes of these 4 miRNAs were primarily enriched in the mitogen activated protein kinase (MAPK) signaling pathway. Therefore, MAPK signaling pathway may serve important roles in MTC progression. In conclusion, the DEMs hsa-miR-1 and hsa-miR-9-5p, and TFs of NF-kappa B1 and MYC may be used as biomarkers for the diagnosis and treatment of MTC.