Gene therapy is a promising therapeutic modality for the treatment of cystic fibrosis (CF). Despite a better understanding of the molecular organization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and mutations resulting in pathophysiological and phenotypic alterations, several forms of treatments including gene therapy have failed to yield clinical success. Major limitations for the delivery of drugs and gene therapy vectors from reaching target cells in CF patients lie in physical and immunological barriers of airway epithelium. Over the last decade, non-viral and viral gene therapy approaches have been tested in preclinical studies and human clinical trials of CF. Outcomes of these studies have helped to identify hurdles that need to be overcome before such approaches can be routinely applied to patients. In addition to the physiological and immunological barriers of airway epithelium, vector transduction is also impaired by the absence or low-abundance of cellular receptors and co-receptors for viral binding and internalization. Thus, the initial enthusiasm for gene replacement therapy for CF following cloning of the CFTR gene dampened, as more limitations were recognized. Research directed towards improving the efficiency of gene transfer technology in CF, is focused on testing of compounds to enhance vector permeability and trafficking, identification and development of vectors which can transduce through alternate pathways, identification of airway epithelium-specific targeting ligands, and the identification of stem cells for combining cell therapy and gene therapy by ex vivo methods. Details provided in this article will give a comprehensive analysis of the prospects and limitations in CF gene therapy using viral and nonviral vectors.