Cationic nanoparticles are powerful tools for delivering nucleic acids to cells in vitro. However, current nanopartcle formulations suffer from inhibition by biological macromolecules, such as serum proteins, and from endosomal retention, limiting their use in vivo. We synthesized a series of novel polyvalent ionizable cationic lipids and tested their ability to deliver mRNA to cells in vitro and in vivo. Using mRNA encoding GFP, we found that delivery was influenced by both headgroup size and degree of unsaturation in the fatty-acid tail. We identified a compound, N1,N4-dilinoleyl-N1,N4-di-(2-hydroxy- 3-aminopropyl)-diaminobutane (DLinDHS or “DLin”), capable of delivering nucleic acids (including mRNA, siRNA, and plasmid DNA) to a variety of cell types and in serum concentrations as high as 100%. DLin/mRNA lipoplexes exhibit ionic strength-dependent particle size and pH-dependent zeta potential. High-resolution, time-resolved fluorescence microscopy using Cy5-labeled mRNA showed that DLin/ mRNA lipoplexes fuse directly with the plasma membrane, bypassing the endosomal-lysosomal system. We demonstrate high levels of protein expression from mRNA delivered with DLin in primary human epidermal keratinocytes, dermal fibroblasts, pluripotent stem cells (iPS cells), PBMCs, dendritic cells, human lung adenocarcinoma cells, and rat embryonic cortical neurons. DLin transfection was equally effective in proliferating cells and contact-inhibited confluent cell monolayers. We also show high-efficiency gene editing of primary human cells using DLin complexed with mRNA encoding both TALENs and NoveSlice, a novel gene-editing endonuclease. Administration of nebulized lipoplexes to rats resulted in protein expression in lung epithelial cells. Intradermal injection of DLin complexed with mRNA encoding a reporter protein in rats and in a human subject yielded localized expression in the dermal layer. In conclusion, we present a novel serum-insensitive nucleic acid delivery system and demonstrate delivery of mRNA in vitro and in vivo, including to the skin and lung.