Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5-/- cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors. © 2013 Landes Bioscience.