Immunotherapy has dramatically changed the treatment landscape for several tumor types. However, the impact of previous radiotherapy (RT) on response to immunotherapy is still unknown. We report the case of a 58-year-old female diagnosed with a squamous anal cell carcinoma previously treated with RT and having a dissociated response to anti-PD1 agent. An extensive analysis of the immune contexture performed on the tissue collected from both previously RT-treated and RT-untreated lesions confirmed differences on immune microenvironment, highlighting the potential impact of radiotherapy on the immune response.