The treatment efficacy of Rituximab on lymphoma as an immunotherapeutic approach is confirmed, but this treatment has limited penetration through the brain micro vessels. Such limitation significantly attenuates the efficacy of systemic administration of this antibody on brain lymphomas. We aimed to confirm that Tadalafil, a long-acting phosphodiesterase type 5 inhibitor, could increase microvascular permeability and Rituximab treatment efficacy in brain lymphomas. We established a mouse brain lymphoma model by planting human-derived lymphoma cell line Raji into brain parenchyma of mice using stereotaxic techniques. After 16 days, 7.0 T magnetic resonance imaging was performed to confirm the presence of the mass. The mice were observed under near-infrared fluorescence after intravenous injection of fluorescence-labeled Rituximab. Evans Blue was used as probe to detect the microvascular permeability of brain lymphomas after Tadalafil administration. Starting from 4 days after implantation, the mice were administered different treatments. Survival analysis of brain lymphoma-loaded mice was performed. Evans Blue detection showed that Tadalafil administration could increase brain vascular permeability in the tumor-bearing group compared with control mice. Rituximab treatment prolonged the survival time of mice compared with the untreated control group (mean 25.75 vs. 20.8 days, p < 0.05). Tadalafil with Rituximab treatment resulted in the longest survival time (29 days, p < 0.05). Rituximab may be a promising therapeutic agent for the treatment of brain lymphoma. Tadalafil can enhance Rituximab treatment efficacy by improving the microvascular permeability in mice brain lymphoma.