Despite significant advances in cystic fibrosis (CF) treatments, a one-time treatment for this life-shortening disease remains elusive. Stable complementation of the disease-causing mutation with a normal copy of the CF transmembrane conductance regulator (CFTR) gene fulfills that goal. Integrating lentiviral vectors are well suited for this purpose, but widespread airway transduction in humans is limited by achievable titers and delivery barriers. Since airway epithelial cells are interconnected through gap junctions, small numbers of cells expressing supraphysiologic levels of CFTR could support sufficient channel function to rescue CF phenotypes. Here, we investigated promoter choice and CFTR codon optimization (coCFTR) as strategies to regulate CFTR expression. We evaluated two promoters—phosphoglycerate kinase (PGK) and elongation factor 1-α (EF1α)—that have been safely used in clinical trials. We also compared the wild-type human CFTR sequence to three alternative coCFTR sequences generated by different algorithms. With the use of the CFTR-mediated anion current in primary human CF airway epithelia to quantify channel expression and function, we determined that EF1α produced greater currents than PGK and identified a coCFTR sequence that conferred significantly increased functional CFTR expression. Optimized promoter and CFTR sequences advance lentiviral vectors toward CF gene therapy clinical trials.