Although under physiological conditions ROS are essential for normal cell signaling, ROS overproduction under oxidative stress conditions may cause damage to biomolecules. Cysteine residues within proteins are often easily oxidized. Reversible cysteine oxidation is used as a mechanism of redox signaling and control of protein function. However, oxidative stress promotes oxidation of protein thiols, leading to the formation of mixed disulfides between proteins and low-molecular-weight thiols. Disulfide reductases such as thioredoxins can reduce disulfide bonds back to free thiols. Acute pancreatitis is currently one of the leading causes of hospital admission for gastrointestinal disorders, and its incidence has increased considerably over the last decade. Acute pancreatitis is an acute inflammatory process of the pancreas, which can affect other organs and systems. Our laboratory described disulfide stress associated with acute inflammatory processes, which is characterized by a decrease in GSH levels as well as protein cysteinylation and gamma-glutamylcysteinylation, without changes in protein glutathionylation. We found an increase in protein cysteinylation levels in TRP14 knockdown cells, and identified 42 proteins whose cysteinylation levels increased in TRP14 deficient cells, such as elongation factor 2 and peroxiredoxin 2. Besides, we demonstrated that cysteinylated Prx2 is inactive, and both TRP14 and Trx1 systems can reduce it and restore its activity. Unexpectedly TRP14 knockout mice showed lower levels of cysteinylated proteins than wild type sham mice in pancreas under basal conditions. However, upon acute pancreatitis a marked increase in protein cysteinylation levels together with cystine levels was observed in TRP14 knockout mice, while there was a decrease in protein cysteinylation levels in wild type mice. Protein gamma-glutamylcysteinylation also increased in TRP14 knockout mice with pancreatitis as well, but without changes in protein glutathionylation. Furthermore, the transsulfuration pathway and GSH synthesis were both induced in TRP14 knockout mice with pancreatitis avoiding GSH depletion. The mRNA expression of Trx1, TrxR1, Gr, as well as other NRF2 targets such as Nqo-1, Gclc, and Ho1 increased in pancreas from wild type mice upon acute pancreatitis, with further increases in pancreas from TRP14 knockout mice with pancreatitis correlated with nuclear translocation of NRF2. At the histological level, TRP14-deficient mice showed less tissue edema and inflammatory infiltrate in pancreas upon pancreatitis induction. Decreased pancreatic MPO activity was also observed in this group. Therefore, TRP14 and thioredoxin 1 exhibit de-cysteinylation activity, and TRP14 deficiency induces protein cysteinylation and gamma-glutamylcysteinylation, NRF2 activation as well as up-regulation of the transsulfuration pathway and GSH synthesis upon acute pancreatitis.