MicroRNAs (miRNAs) are increasingly recognized as oncogenes or tumor suppressors in colorectal cancer (CRC). The aim of this study was to explore the expression and functions of miR-340-5p in CRC.The expression of miR-340-5p in CRC tissues and cell lines was detected by quantitative RT-PCR. Associations of miR-340-5p expression with clinicopathological factors and overall survival (OS) and progression-free survival (PFS) were statistically evaluated. Luciferase assay, RT-PCR, and Western blot were performed to verify the precise target of miR-340-5p. MTT assay, colony formation and transwell assay were performed to determine the proliferation, migration and invasion, respectively.Our results showed that miR-340-5p was significantly down-regulated in CRC tissues and cell lines, and was associated with histological grade (p=0.020), lymph nodes metastasis (p=0.003) and TNM stage (p=0.007). Furthermore, Kaplan-Meier and log-rank tests revealed that patients with low expression of miR-340-5p had a shorter OS (p=0.0110) and PFS (p=0.0032) than those with high expression of miR-340-5p. We further validated Annexin A3 (ANXA3) was a direct target of miR-340-5p in CRC. The functional assay showed that up-regulation of miR-340-5p or down-regulation of ANXA3 can both inhibit CRC cell proliferation, migration, and invasion. Besides, the re-expression of ANXA3 reversed the miR-340-5p induced suppression of cell proliferation, migration and invasion.Our data demonstrated that miR-340-5p exerted its tumor-suppressive function by directly targeting ANXA3 in CRC, suggesting that miR-340-5p might represent a novel prognostic biomarker and therapeutic target for CRC.