The PDZ domain-containing protein CAL mediates lysosomal trafficking and degradation of CFTR. Here we demonstrate the involvement of a CAL-binding SNARE protein syntaxin 6 (STX6) in this process. Overexpression of STX6, which colocalizes and coimmunoprecipitates with CAL, dramatically reduces the steady-state level and stability of CFTR. Conversely, overexpression of a STX6 dominant-negative mutant increases CFTR. Silencing endogenous STX6 increases CFTR but has no effect on DeltaTRL-CFTR, which cannot bind to CAL. Silencing CAL eliminates the effect of STX6 on CFTR. Both results suggest a dependence of CAL on STX6 function. Consistent with its Golgi localization, STX6 does not bind to ER-localized DeltaF508-CFTR. Silencing STX6 has no effect on DeltaF508-CFTR expression. However, overexpression of STX6 coimmunoprecipitates with and reduces temperature-rescued DeltaF508-CFTR that escapes ER degradation. Conversely, silencing STX6 enhances the effect of low temperature in rescuing DeltaF508-CFTR. Finally, in human bronchial epithelial cells, silencing endogenous STX6 leads to increases in protein levels and Cl(-) currents of both wild-type and temperature-rescued CFTR. We have identified STX6 as a new component of the CAL complex that regulates the abundance and function of CFTR at the post-ER level. Our results suggest a therapeutic role of STX6 in enhancing rescued DeltaF508-CFTR.