This study aimed to evaluate the effects of administration of sodium humate (HNa) and glutamine (Gln) on growth performance, diarrhoea incidence, serum parameters, and faecal microflora of pre-weaned Holstein calves. In a 57-day experiment, 28 healthy newborn female calves were randomly allocated to four treatment groups: (1) CON (control); (2) HNa (basal diet + 5% HNa); (3) Gln (basal diet + 1% Gln); and (4) HNa + Gln (basal diet + 5% HNa + 1% Gln). The calves in the CON group were fed with basal diet.HNa and Gln were alone or together mixed with milk (Days 1-20) or milk replacer (Days 21-57) and orally administered to each calf. The results indicated that calves combined supplemented with HNa and Gln had a higher average daily gain at 0-21 days, 21-57 days, and 0-57 days, and starter intake at 21-57 days and 0-57 days (p < 0.05).Compared with the CON group, calves in HNa, Gln, and HNa + Gln groups showed lower faecal scores and diarrhoea incidence at 0-21 days and 0-57 days (p < 0.05). Combined administration of HNa and Gln increased the concentration of IgG and IgA, activities of glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) but decreased the concentration of diamine oxidase (DAO), D-lactic acid (D-lac), TNF-α, and malondialdehyde (MDA) in the serum of calves compared with the CON group throughout the entire period (p < 0.05). Furthermore, the abundances of Bifidobacterium and Lactobacillus were increased but the Escherichia coli was decreased in faecal grab samples of HNa + Gln group calves in comparison with the CON group (p < 0.05). In conclusion, combined administration of HNa and Gln effectively improved the growth performance, antioxidant and immune status, and intestinal beneficial bacteria, and further reduced the diarrhoea incidence of the pre-weaned calves.