Our aim is to study the effects of varying the two acyl moieties in synthesized N4,N9-diacyl spermines on siRNA formulations and their delivery efficiency in cell lines. Six novel asymmetrical lipopolyamines, [N4-cholesteryloxy-3-carbonyl-N9-oleoyl-, N4-decanoyl-N9-oleoyl-, N4-decanoyl-N9-stearoyl-, N4-lithocholoyl-N9-oleoyl-, N4-myristoleoyl-N9-myristoyl-, and N4-oleoyl-N9-stearoyl]-1,12-diamino-4,9-diazadodecane, were assessed for their abilities to bind to siRNA, studied using a RiboGreen intercalation assay, and to form nanoparticles. Their siRNA delivery efficiencies were quantified in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA) using a fluorescein-tagged siRNA, and compared with formulations of N4,N9-dioleoyl-1,12-diamino-4,9-diazadodecane and of a leading transfecting agent, TransIT-TKO. Transfection was measured in terms of siRNA delivery and silencing of EGFP reporter gene in HeLa cells. By incorporating two different acyl moieties, changing their length and oxidation level in a controlled manner, we show efficient fluorescein-tagged siRNA formulation, delivery, and knock-down of EGFP reporter gene. N4-Oleoyl-N9-stearoyl spermine and N4-myristoleoyl-N9-myristoyl spermine are effective siRNA delivery vectors typically resulting in 89% cell delivery and gene silencing to 34% in the presence of serum, comparable with the results obtained with TransIT-TKO; adding a second lipid chain is better than incorporating a steroid moiety.