Urinary excretion of purine derivatives (PDs) is used to estimate the microbial N supply to sheep. The objective of this study was to determine the effect of water restriction following feeding on microbial N supply, and urinary excretion of PDs in Corriedale ewes under heat stress condition. Nine Corriedale ewes (average BW = 45.0 ± 4.5 kg) were fed diets based on maintenance requirements individually in metabolic crates. Sheep were assigned to 3 treatment groups according to a 3 × 3 Latin square design for 3 periods of 21 d duration each (9 sheep per treatment). Treatments were free access to water (FAW), 2h water restriction (2hWR), and 3h water restriction (3hWR) following feeding. No differences were found in body weight gain among all treatment groups (P > 0.05). Daily water intake decreased linearly as water restriction time following feeding increased (P < 0.05). Urine weight and volume were higher (P < 0.05) in FAW than restricted groups (P > 0.05). Water restriction following feeding decreased the urinary N (g/d) and urinary N as a percentage of N intakes (P < 0.05); however, fecal N (g/d) and fecal N as a percentage of N intake were not affected by water restriction following feeding (P > 0.05). Allantoin concentration tended to be higher (P = 0.07) in FAW group than 2hWR and 3hWR groups while no differences (P > 0.05) were observed in uric acid, xanthine + hypoxanthine and creatinine concentrations. Sum of PDs tended to be higher in FAW (P = 0.08) but water restriction following feeding reduced total PDs excretion compared with the FAW group (P < 0.01). It is concluded that water restriction following feeding for less than 3 h improved microbial N supply in ewes under heat stress condition.