MicroRNAs are a class of small, noncoding RNAs that function as critical regulators of gene expression by targeting mRNAs for translational repression or degradation. In this study, we demonstrate that expression of microRNA-124 (miR-124) is significantly downregulated in colorectal cancer tissues and cell lines, compared to the matched adjacent tissues. We identified and confirmed inhibitor of apoptosis-stimulating protein of p53 (iASPP) as a novel, direct target of miR-124 using target prediction algorithms and luciferase reporter gene assays. Overexpression of miR-124 suppressed iASPP protein expression, upregulated expression of the downstream signaling molecule nuclear factor-kappa B (NF- κ B), and attenuated cell viability, proliferation, and colony formation in SW480 and HT-29 colorectal cancer cells in vitro. Forced overexpression of iASPP partly rescued the inhibitory effect of miR-124 on SW480 and HT29 cell proliferation. Taken together, these findings shed light on the role and mechanism of action of miR-124, indicate that the miR-124/iASPP axis can regulate the proliferation of colorectal cancer cells, and suggest that miR-124 may serve as a potential therapeutic target for colorectal cancer.