Solid lipid-polymer hybrid nanocarrier (LPN) was previously reported to achieve high siRNA transfection efficiency and induce sustained RNAi-based chemosensitizing effect at cellular level. In this study, our objectives were to evaluate the in vivo biodistribution of LPNs in a prostate cancer model and determine the factors that potentially affect tumor penetration by LPNs. The LPN formulation with the highest transfection efficiency (64%) and stability was selected for the study. Mice bearing tumors of PC-3M cells were treated with LPNs labeled with IR780 or AF647-siRNA. Near infrared imaging showed that LPNs achieved favorable in vivo biodistribution with high tumor/low organ ratios. LPN accumulation was also observed in liver metastatic tissue. Result of extravasation study confirmed that encapsulated siRNA molecules were able to escape into the tumor tissue at the extravascular area. When LPN levels in large (volume > 750 mm3) and small (<500 mm3) tumors were compared, no significant difference was observed. However, both docetaxel pretreatment (72 h before LPN) and concurrent docetaxel treatment significantly enhanced the tumor LPN levels by 3.9- and 3.1-fold, respectively (both p < 0.01). In conclusion, LPN is a promising carrier system to deliver RNAi therapy to solid malignancies that also receive chemotherapy.