Targeting brain tumor largely depends on the chemotherapeutic agent uptake by the cancerous cells. The uptake further depends on its dosage form, route of administration and surface modifications. In the present study, temozolomide (TMZ) loaded into poly lactic acid (PLA) nanoparticles was employed to evaluate the efficacy against C6 glioma cells. Solvent evaporation method was used to fabricate PLA nanoparticles. Particle size determination, zeta potential and diffusion studies were performed for the plain drug and drug-loaded nanoparticulate delivery system. The functional assessment was performed using in vitro studies, such as MTT, clonogenic and wound scratch assays respectively. The cellular uptake of formulated nanoparticles was investigated using flow cytometry and confocal microscopy. The results indicate that the action of TMZ against C6 glioma is sustained when loaded into nanoparticles. This observation is of clinical significance, as it would avoid repeated administration of the drug as well as minimising the side effects and toxicity.