In the current study, the 5-HT4R agonist RS67333 inhibited diabetes-induced upregulation of MLCK, ROCK1, and p-MLC in the colon, but the agonist failed to produce such an effect when5-HT4 R was knocked out, indicating that 5-HT4R-induced activation of PKA may lead to inhibition of MLCK/ROCK1 expression and downstream phosphorylation of MLC. Here, HTR4 is linked to diabetes mellitus.