A previous study has revealed that proton beam irradiation affects cell migration in MDA-MB-231 human breast cancer cells. Cyclooxygenase-2 (COX-2) and matrix metalloproteinase‑9 (MMP-9) are highly expressed in various cancers, such as colon, lung and breast cancer, and enhance cell migration and metastasis in vitro and in vivo. In the present study, the effects of proton beam irradiation on COX-2 and MMP-9 expression levels in MDA-MB‑231 human breast cancer cells were investigated, along with the signaling pathway involved in the proton beam irradiation‑mediated antimetastatic effect. The results revealed that 12-O-tetradecanoylphorbol-13‑acetate‑induced increases in COX-2 and MMP-9 expression levels were reversed by proton beam irradiation in a dose-dependent manner. In addition, proton beam irradiation inhibited phosphorylation of protein kinase B (also known as Akt) and nuclear factor-κB (NF-κB), which are activated by phosphoinositide 3-kinase (PI3K) stimulation. MMP-9 and COX-2 expression levels are regulated by PI3K/Akt and/or protein kinase C/mitogen-activated protein kinase signaling pathways that enhance NF-κB and activator protein-1 transcriptional activities. Therefore, the results suggest that proton beam irradiation inhibited the cancer cell growth and metastasis associated with COX-2 and MMP-9 expression in MDA-MB‑231 human breast cancer cells, and that the antimetastatic effect of proton beam irradiation is achieved by the suppression of NF-κB phosphorylation via inhibition of Akt activation.