Aflatoxin (AF) mitigation strategies have been studied to alleviate the AF effects on dairy cattle performance. However, limited research is available on the effects of trace mineral supplementation during an AF challenge. The objective of this study was to determine the performance 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 average body weight (BW) in response to an AF challenge. Fifty-eight Holstein cows [BW (mean ± SD) = 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 dry matter intake (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. Milk was sampled 3 times daily from d 56 to 63. Two treatment orthogonal contrasts, CONT1 (NEG vs. POS) and CONT2 (POS vs. MM), were made. Cows in NEG had lower (P < 0.001) AF excretion in milk (0.0 µg/d) and higher (P = 0.02) 3.5% fat-corrected milk (32.1 kg/d) compared with cows in POS (16.7 µg/d and 28.6 kg/d, respectively). Protein yield tended to be higher (P = 0.10) for cows in CON (1.13 kg/d) compared with cows in POS (1.07 kg/d). Feed efficiencies (3.5% FCM/DMI, ECM/DMI, and Milk/DMI) were higher (P = 0.02) for cows in NEG (1.42, 1.46, and 1.45, respectively) than cows in POS (1.16, 1.18, and 1.22, respectively). Cows in POS had higher (P = 0.03) milk urea nitrogen (14.3 mg/dL) than cows in MM (13.3 mg/dL). In conclusion, subcutaneous supplementation of trace minerals neither hindered nor improved performance response when an AF challenge was present.