The risk of disease increases for post-weaned calves when they are transitioned from individual housing to group housing (commingling). Therefore, this study was conducted to determine if prebiotic supplementation of mannan-oligosaccharide (MOS) and β-glucan (BG) would assist calves in the transition from individual hutches to groups of 3. Feed intake, body weight gain, in vivo adaptive immune responses, and ex vivo neutrophil responses were measured from 60, weaned Holstein heifer calves (age 52 ± 4.0 SD d; 83 ± 14.7 SD kg). One week before commingling (−1 wk), calves were randomly assigned to either a daily bolus dose of oral prebiotics (3 g; 10% MOS, 18% BG) dissolved in 15 mL of molasses or control (15 mL molasses only) for 7 weeks. Daily DMI was collected and calves were weighed weekly. Whole blood was collected via jugular venipuncture on wk −1, 1, 2, and 6 relative to commingling. In addition, all calves were administered an innocuous protein injection, ovalbumin (OVA; subQ; 0.5 mg/mL), at commingling and 4 weeks after commingling. All blood samples were measured via flow-cytometry for peripheral neutrophil phagocytosis (PG) and oxidative burst (OB) responses to heat-killed E. coli (8739). Plasma OVA-specific IgG and IgA were measured at 2 weeks after each OVA injection. Two weeks after commingling, prebiotic-treated calves had neutrophils with greater OB intensity than control calves (P = 0.005). Prebiotic-calves also had greater primary IgA (P < 0.01) and IgG (P = 0.04) responses to OVA than control calves, as well as greater secondary IgA response (P < 0.01) than control calves. One week after commingling, prebiotic-calves had greater ADG (1.07 vs. 0.90 ± 0.059 kg/d; P = 0.04) and as well as lower F:G (2.61 vs. 3.60 ± 0.204; P = 0.001) than control calves. Prebiotic supplements enhanced innate and adaptive immune measures and performance during the commingling phase. This supplementation may help reduce the risk of disease and improve vaccination response during commingling in post-weaned dairy heifers.