FK506-binding protein 38 (FKBP38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, associates with nascent plasma membrane ion channels in the endoplasmic reticulum (ER). It promotes the maturation of the human ether-à-go-go-related gene (HERG) potassium channel and maintains the steady state level of the cystic fibrosis transmembrane conductance regulator (CFTR), but the underlying mechanisms remain unclear. Using a combination of steady state and pulse-chase analyses, we show that FKBP38 knockdown increases protein synthesis but inhibits the post-translational folding of CFTR, leading to reduced steady state levels of CFTR in the ER, decreased processing, and impaired cell surface functional expression in Calu-3 human airway epithelial cells. The membrane anchorage of FKBP38 is necessary for the inhibition of protein synthesis but not for CFTR post-translational folding. In contrast, the peptidylprolyl cis/trans isomerase active site is utilized to promote CFTR post-translational folding but is not important for regulation of protein synthesis. Uncoupling FKBP38 from Hsp90 by substituting a conserved lysine in the TPR domain modestly enhances CFTR maturation and further reduces its synthesis. Removing the N-terminal glutamate-rich domain (ERD) slightly enhances CFTR synthesis but reduces its maturation, suggesting that the ERD contributes to FKBP38 biological activities. Our data support a dual role for FKBP38 in regulating CFTR synthesis and post-translational folding. In contrast to earlier prediction but consistent with in vitro enzymological studies, FKBP38 peptidylprolyl cis/trans isomerase plays an important role in membrane protein biogenesis on the cytoplasmic side of the ER membrane, whose activity is negatively regulated by Hsp90 through the TPR domain.