Nonviral vectors continue to be attractive alternatives to viruses due to their low toxicity and immunogenicity, lack of pathogenicity, and ease of pharmacologic production. However, nonviral vectors also continue to suffer from relatively low levels of gene transfer compared to viruses, thus the drive to improve these vectors continues. Many studies on vector-cell interactions have reported that nonviral vectors bind and enter cells efficiently, but yield low gene expression, thus directing our attention to the intracellular trafficking of these vectors to understand where the obstacles occur. Here, we will review nonviral vector trafficking pathways, which will be considered here as the steps from cell binding to nuclear delivery. Studies on the intracellular trafficking of nonviral vectors has given us valuable insights into the barriers these vectors must overcome to mediate efficient gene transfer. Importantly, we will highlight the different approaches used by researchers to overcome certain trafficking barriers to gene transfer, many of which incorporate components from biological systems that have naturally evolved the capacity to overcome such obstacles. The tools used to study trafficking pathways will also be discussed.