Melatonin is predominately produced and secreted by the pineal gland, and inhibits cell growth in various cancer cell lines such as colorectal cancer. However, the precise mechanisms involved have not been fully elucidated. In the present study, the potential molecular mechanism underlying the efficacy of melatonin on migration in RKO colon cancer cells was investigated. The effects of melatonin and H‑1152, a selective inhibitor of Rho‑associated protein kinase (ROCK), on the migration of RKO cells were analyzed by an in vitro wound healing assay. The localization of zonula occludens‑1 (ZO‑1) and occludin were observed by immunofluorescence. Reverse transcription‑quantitative polymerase chain reaction (qPCR) was performed to analyze the relative mRNA levels of ROCK, ZO‑1 and occludin. In addition, western blot analysis was implemented to examine the expression of ROCK, phospho (p)‑myosin phosphatase targeting subunit 1 (MYPT1), p‑myosin light chains (MLC) and p‑p38. The results revealed that the expression levels of ROCK2, p‑MYPT1 and p‑MLC in RKO cells were decreased, and the membrane protein expression of ZO‑1 and occludin increased when the cells were treated with melatonin. qPCR demonstrated that melatonin downregulated ROCK2 gene expression, and upregulated the expression of the ZO‑1 and occludin genes. The levels of ZO‑1 and occludin localized in the tight junctions were markedly increased in the immunofluorescence assay. In addition, the phosphorylation levels of p38 were reduced when the cells were treated with melatonin, and treatment with H‑1152 downregulated p38 phosphorylation. The results indicated that melatonin may inhibit the migration of RKO colon cancer cells by downregulating ROCK expression via the p38/mitogen‑activated protein kinase signaling pathway.