The purpose of this study is to evaluate the in vivo efficacy, unwanted toxicity and loco-regional distribution of a doxorubicin-loaded polymer-lipid hybrid nanoparticle (Dox-PLN) formulation in a murine solid tumor model after intratumoral injection. Dox-PLN were prepared by dispersing Dox in stearic acid and tristearin, with subsequent addition of a novel anionic polymer HPESO (hydrolyzed polymer of epoxidized soybean oil) to enhance the drug incorporation in the lipids. Solid tumors were obtained by injecting EMT6 mouse mammary cancer cells intramuscularly into the hind legs of BALB/c mice. Dox-PLN, blank PLN or surfactant formulations were injected intratumorally (IT) when tumors reached approximately 0.3 g. In vivo efficacy of treatment was measured by tumor growth delay (TGD), defined as the delay in time for the tumor to grow to 1.13 g relative to the untreated control. Signs of unwanted drug toxicity, the histology and morphology of tumor and heart tissues, and the IT distribution of Dox-PLN after IT treatment were examined or monitored. IT-administered Dox-PLN resulted in 70% and 100% TGD (p<0.01) for Dox doses of 0.1 and 0.2 mg, respectively. Dox-PLN treated tumors developed substantially larger central necrotic regions than the untreated tumors, with Dox-PLN residues extensively distributed among the dead cell debris, suggesting that the anticancer effect of Dox-PLN was mainly a combined result of IT nanoparticle distribution and short-ranged, sustained drug release. Except for two of fifteen mice receiving the higher 0.2 mg Dox dose showing transient fur-roughing, all Dox-PLN treated mice showed no signs of toxicity. The present study demonstrates that Dox-PLN possess significant in vivo cytotoxic activity against solid tumors with minimal systemic toxicity. IT administered Dox-PLN have the potential to improve the therapeutic index of loco-regional solid tumor chemotherapy.