Proton radiotherapy has been established as a highly effective modality used in the local control of tumor growth. Although proton radiotherapy is used worldwide to treat several types of cancer clinically with great success due to superior targeting and energy deposition, the detailed regulatory mechanisms underlying the functions of proton radiation are not yet well understood. Accordingly, in the present study, to assess the effects of proton beam on integrin-mediated signaling pathways, we investigated the expression of integrins related to tumor progression and integrin trafficking, and key molecules related to cell adhesion, as well as examining phosphorylation of signaling molecules involved in integrin-mediated signaling pathways. Proton beam irradiation inhibited the increase in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced integrin β1 protein expression and the gene expression of members of the integrin family, such as α5β1, α6β4, αvβ3, and αvβ6 in human colorectal adenocarcinoma HT-29 cells. Simultaneously, the gene expression of cell adhesion molecules, such as FAK and CDH1, and integrin trafficking regulators, such as RAB4, RAB11, and HAX1, was decreased by proton beam irradiation. Moreover, proton beam irradiation decreased the phosphorylation of key molecules involved in integrin signaling, such as FAK, Src, and p130Cas, as well as PKC and MAPK, which are known as promoters of cell migration, while increased the phosphorylation of AMPK and the gene expression of Rab IP4 involved in the inhibition of cell adhesion and cell spreading. Taken together, our findings suggest that proton beam irradiation can inhibit metastatic potential, including cell adhesion and migration, by modulating the gene expression of molecules involved in integrin trafficking and integrin-mediated signaling, which are necessary for tumor progression.