MicroRNA-9 (miR-9) has been reported to play a suppressive or promoting role according to cancer type. In this study, we investigated the effects of anoctamin-1 (ANO1) and miR-9 on colorectal cancer (CRC) cell proliferation, migration, and invasion and determined the underlying molecular mechanisms. Thirty-two paired CRC tissues and adjacent normal tissues were analyzed for ANO1 expression using quantitative real-time PCR (qRT-PCR). HCT116 cells were transiently transfected with miR-9 mimic, miR-9 inhibitor, or si-ANO1. Cell proliferation was determined by MTT, and flow cytometric analysis, while cell migration and invasion were assayed by trans-well migration and invasion assay in HCT116 cells. ANO1 was validated as a target of miR-9 using luciferase reporter assay and bioinformatics algorithms. We found that ANO1 expression was up-regulated in CRC tissues compared with adjacent normal tissues. ANO1 expression was associated with advanced tumor stage and lymph node metastasis, and there was an inverse relationship between miR-9 and ANO1 mRNA expression in CRC specimens, but no significant difference was found between miR-9 and ANO1 expression. ANO1 is a direct target of miR-9, and overexpression of miR-9 suppressed both mRNA and protein expression of ANO1 and inhibited cell proliferation, migration, and invasion of HCT116 cells. We also showed that overexpression of miR-9 suppressed expression of p-AKT, cyclin D1, and p-ERK in HCT116 cells. We conclude that miR-9 inhibits CRC cell proliferation, migration, and invasion by directly targeting ANO1, and miR-9/ANO1 could be a potential therapeutic target for CRC.