〔Abstract〕 Objective To investigate the effect of homocysteine(Hcy) on the permeability of Caco-2 cells and its mechanism. Methods Caco-2 cells were divided into blank control group,treated with different concentrations of Hcy(10, 20, 50 μmol/L), treated with Hcy + different signaling transduction inhibitors(ERK inhibitor PD98059, MLCK inhibitor ML-7, P38 MAPK inhibitor SB203580, Ca2+/Calmodulin inhibitor KN62, Rho inhibitor Y27632 and PKC inhibitor Staurosporine). The permeability of Caco-2 cells was detected by transepithelial electrical resistance(TEER) and fluorescein isothiocyanate(FITC). The expression levels of MEK, ERK, MLCK, p-ERK, p-MLCK, ZO-1, Occludin, Claudin-1 and Claudin-2 in Caco-2 cells were detected by Western blot. Results Compared with the blank control group, the permeability of Caco-2 cells pretreated with 50 μmol/L Hcy had the faster change. The expression of Claudin-1, Occludin and ZO-1 decreased, while the expression of MEK, ERK, MLCK, p-ERK, p-MLCK and Claudin-2 increased. Compared with Hcy group, pretreatment with ML-7 and PD98059 could reduce the permeability alteration of Caco-2 cells induced by Hcy, which resulted in decreased expression of MEK, ERK, MLCK, p-ERK, p-MLCK, Claudin-2 while increased expression of Claudin-1, Occludin and ZO-1. Conclusion Hcy may regulate the expression of tight junction-related proteins through MEK-ERK-MLCK signaling pathway and affect the permeability of Caco-2 cells.