Canine spontaneous intracranial tumors bear striking similarities to their human tumor counterparts and have the potential to provide a large animal model system for more realistic validation of novel therapies typically developed in small rodent models. We used spontaneously occurring canine gliomas to investigate the use of convection-enhanced delivery (CED) of liposomal nanoparticles, containing topoisomerase inhibitor CPT-11. To facilitate visualization of intratumoral infusions by real-time magnetic resonance imaging (MRI), we included identically formulated liposomes loaded with Gadoteridol. Real-time MRI defined distribution of infusate within both tumor and normal brain tissues. The most important limiting factor for volume of distribution within tumor tissue was the leakage of infusate into ventricular or subarachnoid spaces. Decreased tumor volume, tumor necrosis, and modulation of tumor phenotype correlated with volume of distribution of infusate (Vd), infusion location, and leakage as determined by real-time MRI and histopathology. This study demonstrates the potential for canine spontaneous gliomas as a model system for the validation and development of novel therapeutic strategies for human brain tumors. Data obtained from infusions monitored in real time in a large, spontaneous tumor may provide information, allowing more accurate prediction and optimization of infusion parameters. Variability in Vd between tumors strongly suggests that real-time imaging should be an essential component of CED therapeutic trials to allow minimization of inappropriate infusions and accurate assessment of clinical outcomes.