The pre-weaning phase is critical for calf development, with long-term implications for health and productivity. This study evaluated the effects of gamma-aminobutyric acid (GABA) supplementation on growth, ruminal fermentation, diarrhoea, and antioxidant capacity in pre-weaned Holstein calves. Ninety male calves were assigned to five GABA dosage groups (0–200 mg/day) from 11 to 75 days of age. Supplementation at 100 mg/day significantly improved average daily gain and body height without affecting diarrhoea incidence. GABA reduced total volatile fatty acids and acetate levels in the rumen, while butyrate showed a dose-dependent response. These findings suggest that 100 mg/day of GABA optimally enhances growth and regulates rumen fermentation, offering a promising strategy for improving calf performance before weaning.