Problems related to the safety and efficacy of gene therapies have checked the enthusiasm once surrounding this field, though it remains a promising approach for the treatment of numerous diseases. Despite the high transfection efficiencies attainable using viral vectors, manufacturing difficulties, safety concerns, and limitations related to targeting and plasmid size have prompted considerable research into the development of non-viral vectors. Non-viral vectors demonstrate low toxicity, low immunogenicity, and ease of manufacture. However, they have not yet achieved the transfection efficiencies displayed by viruses. The inability to explain or predict transfection efficiencies results, in part, from insufficient understanding of the intracellular processes involved in gene delivery. Increasingly, research has been undertaken to probe the processes involved in overcoming the major obstacles to vector-mediated transfection: (1) internalization, (2) intracellular trafficking, (3) escape to the cytosol, (4) nuclear translocation, and (5) gene transcription/expression. This paper reviews and compares the pathways and techniques involved in successful viral and non-viral transfection. In addition, this review provides evidence that non-viral vector development has been pursued successfully thus far, producing systems capable of evading almost all major obstacles to transfection. Evaluating the abilities of non-viral and viral vectors to overcome specific cellular barriers reveals that the greatest advantage of viral vectors may be related to viral DNA, which is transcribed considerably more efficiently than plasmid DNA. Further study in this area should enable the development of non-viral vectors that transfect as efficiently as viral vectors.