This study aimed to assess the impact of betaine supplementation on blood biomarkers and metabolism in dairy cows exposed to humid, hot conditions. Thirty lactating cows from an Egyptian industrial dairy farm were divided into two groups: a control group receiving no betaine (n=15) and a supplemented group (80g betaine/day for four weeks, n=15). Blood samples were collected at three-time points: baseline zero week (0w), two weeks (2w), and four weeks (4w). The levels of cortisol, superoxide dismutase (SOD), malondialdehyde (MDA), albumin, total protein, insulin, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, non-esterified fatty acids (NEFA), and beta-hydroxybutyric acid (BHBA) were measured and compared between the groups using linear t-test and across time points using repeated measure ANOVA. Additionally, milk production was recorded throughout the study period. The results indicated that cortisol levels were significantly lower in the betaine group at 2w and 4w compared to the control group. Urea and ALT levels were consistently more down in the betaine group at all time points. BHBA and NEFA levels showed significant decreases in the betaine group at 4w. SOD level was notably higher in the betaine group at 2w and 4w. Finally, milk yield was improved in the betaine-supplemented cows than in the control group. In conclusion, supplementing dairy cows with betaine positively influences blood metabolites and antioxidant profiles, improving homeostatic response to heat stress and increasing milk production.