Dietary nitrate, from green leaf vegetables, has raised a great deal of interest in the recent few years as the main source of nitric oxide (●NO) in the human gut via nitrate-nitrite-nitric oxide pathway. The physiological significance of this pathway is connected to the non-enzymatic reduction of nitrite to ●NO and other bioactive nitrogen oxides in the stomach and ensued impact in human health and therapeutics. The bioactive nitrogen oxides are acknowledged for their oxidizing and nitrating capacity, modifying the function of lipids and proteins. This study shows a nitrate-dependent nitrating pathway targeting a tight junction protein in the stomach, occludin. This transmembrane protein is located in the most apical position of epithelial cell membrane and exhibits two extracellular loops containing up to 60% of tyrosine residues. Hence, both the chemical structure and location of occludin make it a potential target for nitration. All animal experiments were performed according to the ARRIVE guidelines and the European Community Council Directive for the Care and Use of Laboratory Animals (86/609/ECC). Male Wistar rats (n=5, per group) were used in this study. Inorganic nitrate (10 mM), nitrite (2 mM) or human saliva collected following lettuce ingestion were administered by oral gavage. In order to ensure a completed enterosalivary cycle, the nitrate circulation occurred for 4 hours. After this period, the rats were anesthetized (halothane) and euthanized by cervical dislocation. The stomachs were then collected. Nitrated occludin was detected by immunoprecipitation in the gastric epithelium upon inorganic nitrite administration (p<0.05, through a t-student test). No significant nitration was observed in the case of inorganic nitrate or human saliva. Accordingly, salivary ascorbate, urate and thiocyanate promote nitrite reduction to ●NO in the acidic pH of the stomach diverting the production of nitrated species. Furthermore, different ●NO production rates were observed when using inorganic or salivary nitrite. Using an electrochemical approach, we observed that sodium nitrite produced lower steady state concentrations of ●NO (0.12±0.03 μM) as compared to salivary nitrite (0.37±0.01 μM). These findings indicate that the production of nitrating agents or other stable oxides could be affected by competing reactions at acidic pH. The biological impact of occludin nitration remains elusive. Considering, that the nitrate-nitrite-nitric oxide pathway, by generating ●NO in the gastric lumen, could modulate gut barrier function, the implications for gastrointestinal disorders such as inflammatory bowel disease or other leaky disorders should be further investigated.