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OBJECTIVES: Dietary inorganic nitrate from foods such as red beetroot juice (RBJ) can contribute to nitric oxide (NO) bioavailability through the enterosalivary nitrate-nitrite-NO pathway. A critical step in this pathway is the reduction of nitrate to nitrite by oral bacteria. We investigated the effects of inorganic nitrate supplementation, as RBJ or placebo + potassium nitrate (PBO+NIT), on the oral microbiota, and its relationship with saliva and plasma NO metabolites and vascular endothelial function. METHODS: In a randomized, double-blind, placebo-controlled trial, we measured the abundance of oral nitrate-reducing bacteria in saliva samples from 15 middle-aged/older adults with overweight and obesity using 16 rRNA sequencing. We also assessed the relationship of oral nitrate-reducing bacteria with the physiological responses to acute (4 hours) and chronic (4 weeks) RBJ, PBO+NIT, nitrate-free RBJ, and placebo supplementation via measurement of saliva and plasma nitrate/nitrite (NOx), plasma nitrite levels, and reactive hyperemia index (RHI). RESULTS: A significant decrease in the alpha diversity metric, Pileou's Evenness, was detected after chronic consumption of PBO+NIT (0.69 ± 0.05 at week 0 vs. 0.65 ± 0.05 at week 4; P