For the application of RNA interference (RNAi) in vivo the functional delivery of short interfering RNAs (siRNAs) is still the major obstacle. Therefore, delivery technologies need to be established for the systemic application of RNAi in vivo. Here we report uptake, biodistribution and in vivo efficacy of siRNA molecules formulated into siRNA-lipoplexes. The applied formulation is based on complex formation of positively charged liposomes, a mixture of cationic and fusogenic lipids complexed with the negatively charged siRNA. We determined by fluorescence microscopy the temporal and spatial distribution of fluorescently labeled siRNA-lipoplexes, the body clearance and endothelial cell type specific uptake after single intravenous injection. Furthermore, by using siRNA molecules for targeting endothelia-specifically expressed genes, such as CD31 and Tie2, we were able to demonstrate downregulation of the corresponding mRNA and protein in vivo. Taken together, we show the applicability of this non-viral delivery technology for inducing RNAi in the vasculature of mice after systemic application.