Doxorubicin, an approved drug for cancer therapy, was recently found to be a potent agent to augment adeno-associated virus (AAV)-mediated transgene expression, especially in airway cells. Recombinant AAV type 2 (rAAV-2) has been shown to preferentially transduce neural tissues and is considered as the primary viral vector for the treatments of various neurodegenerative diseases including Parkinson's disease (PD). The goal of this study is to investigate whether doxorubicin can be applied to increase the efficacy of rAAV-2 transduction in the central nervous system. We co-administrated doxorubicin with AV2.luc/EGFP into the rat striatum, a preferred target site for PD gene therapy, and found that doxorubicin augmented rAAV-2 transduction dramatically without significant cytotoxicity and alteration of rAAV-2 tropism. By evaluating the effects of doxorubicin on rAAV-2 transduction in PC12 and MN9D neuronal cells, we found that doxorubicin appeared to promote the nuclear accumulation of rAAV-2, but did not affect viral binding or uptake. Our data suggested that doxorubicin might play an important role in modulating rAAV-2 intracellular trafficking in neuron-like cells. Our study also provided the initial in vivo evidence to facilitate AAV-mediated gene expression in the midbrain with the treatment of doxorubicin.