It is important to obtain structure–activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N4,N9-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N4,N9-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N4,N9-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.