Trace minerals are vital in oxidative stress; however, limited research is available on the effects of trace mineral supplementation during an aflatoxin (AF) challenge. The objective of the study was to determine the immunological effects of 2 subcutaneous supplementations of 15 mg/mL Cu, 5 mg/mL Se, 60 mg/mL Zn, and 10 mg/mL Mn (Multimin 90, Multimin North America, Fort Collins, CO) given at 1 mL/90.7 kg of BW in response to an AF challenge. Fifty-eight Holstein cows [BW (734 ± 60kg); DIM (191 ± 93)] were assigned to 1 of 3 treatments in a randomized complete block design. The experimental period (63 d) was divided into an adaptation phase (d 1 to 56) and a measurement phase (d 57 to 63). From d 57 to 59 cows received an AF challenge, which consisted of 100 μg of AFB1/kg of dietary DMI administered orally via balling gun. Treatments were: saline injection and no AF challenge (NEG), saline injection and AF challenge (POS), and trace mineral injection and AF challenge (MM). Injections were performed subcutaneously on d 1 and 29 at 1 mL/90.7 kg of average BW. Blood was sampled on d 0, 56, 60, and 63, and liver samples were taken on d 0 and 60. Two treatment orthogonal contrasts, CONT1 (NEG vs. POS) and CONT2 (POS vs. MM), were made. Milk urea nitrogen and BUN were higher for cows in POS (14.3 mg/dL and 16.5 mg/dL, respectively) than cows in MM (13.3 mg/dL and 15.8 mg/dL, respectively; P = 0.03 and 0.04, respectively). Liver concentrations of Se and Fe were higher for cows in MM (4.6 and 214.4 µg/kg liver DM, respectively) compared with cows in POS (4.0 and 190.8 µg/kg liver DM, respectively; P = 0.04 and 0.02, respectively). Cows in MM tended to have higher plasma glutathione peroxidase activity (30.2 nmol/min/mL) compared with cows in POS (24.2 nmol/min/mL; P = 0.10). An upregulation of liver GPX1 was observed for cows in POS (1.07 ± 0.05) compared with cows in MM (0.95 ± 0.05; P = 0.01). In conclusion, subcutaneous supplementation of trace minerals induced a positive antioxidant response during an AF challenge.