Background: VEGF is an angiogenic protein that has been studied extensively for its role in benign and pathogenic angiogenesis. VEGF transcription and translation are up-regulated in response to hypoxia, and, paradoxically, also by hyperoxia. Small interference RNA (siRNA) interferes with gene expression by inhibiting translation of protein. Most studies to date have examined the effects of siRNA in vitro, and do not address the problems of systemic administration of siRNA in an in vivo model. Studies that examine siRNA in vivo typically involve the use of methods that are either nonphysiologic, cause an immune response, or are not clinically applicable to humans (e.g., tail vein injection). Purpose: The purpose of this study was to evaluate if a decrease in VEGF protein levels in skeletal muscle could be achieved in vivo, using a systemic route of administration of siRNA that is both clinically applicable and physiologic. Materials and Methods: siRNA targeting VEGF mRNA was complexed with a linear polyethylenimine (PEI). Using a weight-based protocol, male Wistar rats were given a single intraperitoneal injection of the siRNA-PEI complex. A gracilis muscle flap was raised with single vessel inflow and outflow. In a preliminary experiment, a 24-hour interval between injection and tissue harvest was found to provide maximal down-regulation of VEGF protein levels. Subsequent animals were injected 24 hours in advance with the siRNA-PEI complex; gracilis muscle was then harvested and analyzed for VEGF protein levels via Western Blot. Control consisted of animals injected with a nonsilencing siRNA complexed with PEI. VEGF 164 and 120 protein levels were calculated based on a percentage of their respective positive controls and expressed in nanograms. Results were analyzed using ANOVA, with a p value ≤ 0.05 accepted as significant. Results: Maximal down-regulation of VEGF 164 protein levels in gracilis muscle occurred 24 hours after injection. VEGF 164 protein levels were decreased in the VEGF siRNA group by 69% compared to the nonsilencing siRNA group (p < 0.05). There was no statistically significant difference in VEGF 120 levels between the two groups. Conclusion: Small interference RNA can be safely and successfully delivered to distant targets such as skeletal muscle after systemic administration, using intraperitoneal injection of siRNA complexed with a linear polyethylenimine. VEGF 164 levels in skeletal muscle can be down-regulated by almost 70% using this clinically applicable and physiologic route of administration. These data may be useful in future in vivo studies examining siRNA and/or the function of VEGF.