Accumulating evidence has indicated that microRNAs (miRNAs) can act as candidate tumor suppressors. However, the mechanism of miR-27b-3p in colorectal cancer (CRC) is still unknown. In this study, the relative expression levels of miR-27b-3p and LIMK1 were detected by quantitative real-time PCR (qRT-PCR) in CRC and adjacent normal tissues as well as in CRC cell lines. The proliferation, cell cycle and migratory and invasive abilities of SW620 cells were analyzed by CCK-8, flow cytometry, wound healing assays, and Transwell assays, respectively. The regulatory relationships between LIMK1 and miR-27b-3p were detected by qRT-PCR, luciferase reporter gene and Western blot assays. The results showed that the expression levels of miR-27b-3p were downregulated in CRC samples compared with those in adjacent nontumor tissues. Moreover, we found that miR-27b-3p suppressed CRC cell proliferation, cycle and migration and invasion. Additionally, there was an inverse correlation between LIMK1 expression and miR-27b-3p expression in CRC tissues. LIMK1 was identified as a direct target of miR-27b-3p. Our findings indicated that miR-27b-3p inhibited CRC cell proliferation, migration and invasion by targeting LIMK1.