Background <p>We assessed immunotherapy response in a murine melanoma model using multiparametric magnetic resonance imaging (mpMRI) features with <i>ex vivo</i> immunohistochemical validation.</p> Methods <p>Murine melanoma cells (B16-F10) were inoculated into the subcutaneous flank of <i>n</i> = 28 C57BL/6 mice (<i>n</i> = 14 therapy; <i>n</i> = 14 control). Baseline mpMRI was acquired on day 7 at 3 T. The immunotherapy group received three intraperitoneal injections of anti-PD-L1 and anti-CTLA-4 antibodies on days 7, 9, and 11 after inoculation. Controls received a volume equivalent placebo. Follow-up mpMRI was performed on day 12. We assessed tumor volume, diffusion-weighted imaging parameters, including the apparent diffusion coefficient (ADC), and dynamic-contrast-enhanced metrics, including plasma volume and plasma flow. Tumor-infiltrating lymphocytes (TIL; CD8+), cell proliferation (Ki-67), apoptosis (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling, TUNEL), and microvascular density (CD31+) were assessed in a validation cohort of <i>n</i> = 24 animals for time-matched <i>ex vivo</i> validation.</p> Results <p>An increase in tumor volume was observed in both groups (<i>p</i> ≤ 0.004) without difference at follow-up (<i>p</i> = 0.630). A lower ADC value was observed in the immunotherapy group at follow-up (<i>p</i> = 0.001). Immunohistochemistry revealed higher TUNEL values (<i>p</i> &lt; 0.001) and CD8+ TILs (<i>p</i> = 0.048) following immunotherapy, as well as lower tumor cell Ki-67 values (<i>p</i> &lt; 0.001) and microvascular density/CD31+ (<i>p</i> &lt; 0.001).</p> Conclusion <p>Lower tumor ADC, paired with higher intratumoral expression of CD8+ TIL, was observed five days after immunotherapy, suggestive of early immunological response. <i>Ex vivo</i> immunohistochemistry confirmed the antitumoral efficacy of immunotherapy.</p> Relevance statement <p>Compared to tumor size, diffusion-weighted MRI demonstrated potential for early response assessment to immunotherapy in a murine melanoma model, which could reflect changes in the tumor microenvironment and immune cell infiltration.</p> Key Points <p><UnorderedList Mark="Bullet"> <ItemContent> <p>No difference in tumor volume was observed between groups before and after therapy.</p> </ItemContent> <ItemContent> <p>Lower ADC values paired with increased CD8+ TILs were observed following immunotherapy.</p> </ItemContent> <ItemContent> <p><i>Ex vivo</i> immunohistochemistry confirmed antitumoral efficacy of anti-PD-L1 and anti-CTLA-4 immunotherapy.</p> </ItemContent> </UnorderedList></p> Graphical Abstract <p></p>