Background <p>Proton beam therapy is an advanced treatment for patients with unresectable hepatocellular carcinoma. However, evaluating the response to treatment with tumor size alone is insufficient. Herein, we used kinetic [18F]-FDG PET and diffusion-weighted MR imaging to monitor the biological responses to proton beam therapy in hepatocellular carcinoma mice to assess treatment efficacy. Murine BNL HCC cells were orthotopically implanted into the livers of 8-week-old male BALB/c mice, which received 20&#xa0;Gy of the single dose in proton beam therapy. The biological responses to proton beam therapy were assessed on pre-treatment and post-treatment days 1, 3, and 7.</p> Results <p>Compared with the not-receiving proton beam therapy group, the treated group led to an increasing trend in tumor K1 values and constant relative SUVs within 7 days on the dynamic PET imaging. On diffusion-weighted MR imaging, the tumor relative apparent diffusion coefficient values significantly increased post-treatment days 3 and 7. Significantly decreased tumor proliferation, cellular density, and cellular uptake of [18F]-FDG on days 1 and/or 3 post-treatment, with a rebound on day 7, were observed in the dynamic profiling of tumor cells ex vivo and in vitro. Vascular remodeling and elevated macrophage infiltrates in the tumor microenvironment were associated with proton beam therapy. However, there were no significant changes in tumor size between the treated and non-treated groups after treatment until day 7.</p> Conclusions <p>In vivo kinetic [18F]-FDG PET/MRI techniques can provide a feasible means to assess early liver tumor response to proton beam therapy and predict treatment outcomes.</p>