Deregulation of microRNAs is a frequent event in the tumorigenesis and tumor progression. The aim of this study was to investigate the clinical significance and potential role of miR-24-3p expression in colorectal cancer (CRC). The expression level of miR-24-3p was determined in 95 CRC patients who underwent radical resection by quantitative real-time PCR. The associations between miR-24-3p expression and clinicopathological parameters were analyzed. In vitro function assays including cell proliferation, cell migration and invasion were further explored. We found that miR-24-3p was reduced in CRC tissues compared with their corresponding non-cancerous tissues (P < 0.001) and significantly correlated with local invasion (P = 0.002), lymph node metastasis (P = 0.0007) and clinical stage (P < 0.001). Moreover, Kaplan-Meier survival analysis showed that patients with low miR-24-3p level had a significantly poorer prognosis than those with high miR-24-3p level (P < 0.001). Multivariate analysis revealed that miR-24-3p (HR 2.767; 95 % CI 1.203-6.364; P = 0.017) and clinical TNM stage (HR 0.456; 95 % CI 0.212-0.978; P = 0.044) could be independent prognostic indicators for overall survival rates of CRC patients. In addition, functional assays showed that over-expression of miR-24-3p suppressed CRC cell proliferation, cell migration and invasion. miR-24-3p functions as a tumor suppressor in CRC. Down-regulation of miR-24-3p contributes to the development and progression of CRC and may have a potential role in prognosis and therapy.