The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein with anion channel activity that reaches the cell surface via the conventional Golgi-mediated secretion pathway under normal conditions. Interestingly, ER-to-Golgi blockade or ER stress induces alternative GRASP-mediated, Golgi-bypassing unconventional trafficking of wild-type CFTR and the disease-causing ΔF508-CFTR, which has folding and trafficking defects (Gee et al., 2011). However, the molecular mechanisms that underlie the unconventional secretion, and particularly, how the CFTR leaves the ER are unknown. Here, we show that Sec16A, the key regulator of conventional ER-to-Golgi transport, plays a critical role in the ER exit of protein cargos during unconventional secretion. In an initial gene-silencing assay, Sec16A knockdown abolished both the conventional cell-surface trafficking of the Golgi complex-glycosylated WT-CFTR (band C) and the unconventional cell-surface trafficking of the ER core-glycosylated WT-CFTR as well as ΔF508-CFTR (band B) induced by Arf1-Q71L or GRASP55 overepxression. The surface expressed CFTR relative to total lysates value was quantified. The results of multiple experiments are summarized and data are shown as mean ± SEM. Statistical analysis was performed using paired t-tests or with one-way ANOVA. Surface/lysates value of CFTR was reduced by Sec16A silencing (band C of WT-CFTR: 100±0.0 vs. 36.09 ± 10.79 i-Sec16A, p< 0.05; band B of WT-CFTR: 100±0.0 vs. 14.29±5.15 i-Sec16A, p< 0.001; Arf1-Q71L induced ΔF508-CFTR: 100±0.0 vs. 43.45±6.97 i-Sec16A, p< 0.001; GRASP55 overexpression induced ΔF508-CFTR: 100±0.0 vs. 38.31 ± 7.34 i-Sec16A, p< 0.001). Moreover, Sec16A colocalized with GRASP55 in mammalian cells, following stimuli that induce unconventional secretion, such as Arf1 mutant-induced ER-to-Golgi blockade or thapsigargin-induced ER stress. GRASP55 overexpression or ER-to-Golgi blockade relocalized the Sec16A puncta into >80% of the whole cellular area in HeLa cells, whereas only ~20% of the cellular area was Sec16A-positive in control cells. In contrast, subcellular localization of Sec31A, a representative subunit of COPII showed no significant alternation under the conditions of GRASP55 expression or ER-to-Golgi blockade. Collectively, the results suggest that the formation of GRASP-mediated unconventional vesicles is accompanied by the assistance of Sec16A, but is independent of the COPII proteins. In addition, IRE1α depletion reduced unconventional secretion of CFTR, while supplementation with Sec16A rescued the IRE1α depletion-induced reduction in unconventional secretion (ANOVA: p < 0.001), suggesting that IRE1α acts as an upstream signal for ER stress-associated unconventional secretion by modulating Sec16A. These findings highlight a novel function of Sec16A as an essential mediator of ER stress-associated unconventional secretion.