Dietary betaine supplementation can ameliorate physiological responses to heat exposure (HE) in sheep. This experiment measured metabolic responses to glucose (intravenous glucose tolerance, IVGTT), insulin (insulin tolerance test, ITT), and adrenocorticotropic hormone (ACTH) challenges in Merino ewes (n = 36, 39.7 kg) maintained at thermoneutral (TN, 21 °C) or HE (18-43 °C) and supplemented with either 0, 2, or 4 g/day dietary betaine (n = 6 per group). Sheep had ad libitum access to water and were pair-fed such that the intake of the TN sheep mimicked that of the HE sheep. After 21 days of treatment, sheep were fitted with jugular catheters and subjected to consecutive daily challenges (IVGTT, ITT, and ACTH, d 21-23, respectively), followed by skeletal muscle and subcutaneous adipose tissue biopsy collections for gene expression analysis (d 24). The HE-treated sheep had a greater insulin:glucose ratio (p = 0.033), a greater estimated homeostatic model assessment of insulin resistance (HOMAIR; p = 0.029), and a reduced revised quantitative insulin sensitivity check index (RQUICKI; p = 0.015). Sheep fed betaine (2 + 4 g/day) had a greater basal plasma insulin (p = 0.017) and a reduced basal non-esterified fatty acid (NEFA; p = 0.036) concentration, while the RQUICKI was reduced (p = 0.001) in sheep fed betaine. The results suggested that betaine supplementation alters lipid metabolism by potentially improving insulin signaling, although these responses differ between TN and HE conditions. There was no other impact of temperature or dietary treatments on the tissue gene expressions measured. Our results support the notion that betaine, in part, acts to modify lipid metabolism.