Chordomas are rare bone tumors arising from remnants of the notochord. Molecular studies to determine the pathways involved in their pathogenesis and develop better treatments are limited. Alterations in microRNAs (miRNAs) play important roles in cancer. miRNAs are small RNA sequences that affect transcriptional and post-transcriptional regulation of gene expression in most eukaryotic organisms. Studies show that miRNA dysregulation is important for tumor initiation and progression. We compared the expression profile of miRNAs in chordomas to that of healthy nucleus pulposus samples to gain insight into the molecular pathogenesis of chordomas. Results of functional studies on one of the altered miRNAs, miR-31, are presented. The comparison between the miRNA profile of chordoma samples and the profile of normal nucleus pulposus samples suggests dysregulation of 53 miRNAs. Thirty miRNAs were upregulated in our tumor samples, while 23 were downregulated. Notably, hsa-miR-140-3p and hsa-miR-148a were upregulated in most chordomas relative to levels in nucleus pulposus cells. Two other miRNAs, hsa-miR-31 and hsa-miR-222, were downregulated in chordomas compared with the control group. Quantification with real-time polymerase chain reaction confirmed up or downregulation of these miRNAs among all samples. Functional analyses showed that hsa-miR-31 has an apoptotic effect on chordoma cells and downregulates the expression of c-MET and radixin. miRNA profiling showed that hsa-miR-31, hsa-miR-222, hsa-miR-140-3p and hsa-miR-148a are differentially expressed in chordomas compared with healthy nucleus pulposus. Our profiling may be the first step toward delineating the differential regulation of cancer-related genes in chordomas, helping to reveal the mechanisms of initiation and progression.