ABSTRACT Transgenic corn and cotton that express Cry and Vip insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt), have been valuable tools for the management of lepidopteran pests. In 2019, a Vip3Aa-resistant strain of Helicoverpa zea (CEW-Vip-RR) was isolated from F 2 screens of field populations in Texas. Characterizing the resistance mechanism in this strain is important for predicting the sustained efficacy of current commercial Bt traits and guiding the development of future transgenic traits. Resistance to insecticidal proteins in Bt traits is commonly associated with reduced toxin binding, with the exception of Vip3Aa resistance being associated to altered proteolytic processing in the insect host gut. Therefore, Vip3Aa protoxin processing was tested by incubation with midgut fluids from CEW-Vip-RR relative to a susceptible strain (CEW-SS). Finding no significant processing differences, alterations in Vip3Aa binding were tested by comparing binding of radiolabeled and biotinylated Vip3Aa toxin to midgut brush border membrane vesicles (BBMV) from CEW-Vip-RR and CEW-SS larvae. Specific Vip3Aa binding to CEW-Vip-RR BBMV in these experiments was consistently reduced when compared with CEW-SS BBMV. These results support that an altered Vip3Aa-receptor is associated with resistance in CEW-Vip-RR. Understanding this resistance mechanism could have important implications for resistance management decisions, considering widespread Cry1 and Cry2 resistance in H. zea populations. IMPORTANCE Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant-incorporated protectant in Bt crops that consistently provides excellent control of H. zea . The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.