Heat exposure (HE) results in decreased production in ruminant species and betaine is proposed as a dietary mitigation method. Merino ewes ( n = 36, 40 kg, n = 6 per group) were maintained at thermoneutral (TN, n = 18, 21 °C) or cyclical HE ( n = 18, 18–43 °C) conditions for 21 days, and supplemented with either 0 (control), 2 or 4 g betaine/day. Sheep had ad libitum access to water and were pair fed such that intake of sheep on the TN treatment matched that of HE animals. Heart rate (HR), respiration rate (RR), rectal (T R ) and skin temperatures (T S ) were measured 3 times daily (0900 h, 1300 h, 1700 h). Plasma samples were obtained on 8 days for glucose and NEFA analysis. The HE treatment increased T R by 0.7 °C (40.1 vs. 39.4 °C for HE and TN respectively p < 0.001), T S by +1.8 °C (39.3 vs. 37.5 °C, p < 0.001) and RR by +46 breaths/min (133 vs. 87 breaths/min, p < 0.001) compared to TN. The 2 g betaine/day treatment decreased T R (39.8, 39.6 and 39.8 °C, p < 0.001), T S (38.7, 38.0 and 38.5 °C, p < 0.001) and RR (114, 102 and 116 breaths/min for control, 2 and 4 g betaine/day, p < 0.001) compared to control. Betaine supplementation decreased plasma NEFA concentrations by ~25 μM (80, 55 and 54 μmol/L for 0, 2 and 4 g/day respectively, p = 0.05). These data indicate that dietary betaine supplementation at 2 g betaine/day provides improvements in physiological responses typical of ewes exposed to heat stress and may be a beneficial supplement for the management of sheep during summer.