In order to find out the effect of betaine supplementation on physiological (rectal temperature, skin temperature, respiration rate and pulse rate) responses, morphological (body length, heart girth and height at withers) parameters, stress markers (cortisol and NEFA levels), growth hormone, dry matter intake (DMI) and body weight of buffalo heifers during hot humid season under field conditions, 14 buffalo heifers were selected and equally divided into 2 groups, i.e. control and treatment (supplemented betaine @ 25 g/animal/day over the farmers practice). Blood samples were collected at fortnightly interval from both the groups and analyzed for stress markers. THI was calculated for measuring the stress levels on buffalo heifers, which remained 􀀡76 indicating severe stress. Physiological responses were significantly lower in treatment group than control. Morphological parameters, body weight and DMI were higher in treatment than control. Plasma cortisol and NEFA levels were significantly lower, whereas plasma growth hormone was significantly higher in treatment group compared to control. ADG was significantly higher in treatment group compared to control. Positive correlation was observed between THI and stress markers, physiological responses and negatively correlated with ADG, DMI and growth hormone. It can be concluded that betaine acted as a potent growth promoter by lowering the levels of stress markers and enhancing the ADG and DMI of buffalo heifers.