Recent studies have explored combining MDM2 antagonists with immunotherapy. Here, we assessed the effect of anti-PD-1 and idasanutlin co-treatment in a syngeneic CT26/BALB/c mouse model. Prior to in vivo testing, idasanutlin’s activity was evaluated in three murine cell lines (B16-F10, CT26, MC38), revealing varied responses linked to p53 status. Compared to human U-2 OS cells, p53-wild-type mouse cells showed weaker but specific responses, including increased p53/p21 expression, reduced viability, and cell cycle arrest. Due to limited efficacy in murine cells, idasanutlin was tested at three doses in vivo. Surprisingly, the lowest dose (50 mg/kg) produced the highest synergistic effect with anti-PD-1 treatment, significantly reducing tumor size. Higher doses (100 and 200 mg/kg) and anti-PD-1 monotherapy provided either no or only limited tumor size reduction. High doses of idasanutlin decreased the proliferation of T cells and depleted T cell numbers within the lymphocyte population, as we show in vitro and in vivo. These findings suggest that low doses of MDM2 antagonists may enhance immunotherapy, while higher doses could interfere with immunotherapy by p53-mediated cell cycle arrest and decreasing the proliferation of the immune cells.