Cereal rye (Secale cereale L.) cover crop can be an effective non-chemical tool for waterhemp [Amaranthus tuberculatus [Moq.] Sauer] suppression in crop production. Previous studies have evaluated A. tuberculatus suppression by cereal rye as part of weed management programs but have not investigated the underlying mechanism of suppression by the cover crop. This study aimed to investigate the effect of cereal rye biomass on A. tuberculatus emergence and development, and on soil environmental parameters (temperature, moisture, and light transmittance) that are key triggers of A. tuberculatus germination to elucidate the mechanism of suppression by the cover crop. A dose-response study was conducted under field conditions in Brooklyn and Janesville, WI, from 2021 to 2023. Cereal rye biomass from a fall-planted field was harvested at anthesis in the spring and dried to constant weight at 60 C to provide 0.0, 0.6, 1.2, 2.4, 4.8, 7.2, 9.6, and 12.0 Mg ha-1 of dry biomass which was evenly distributed over 1.9 m-2 plots. Increasing cereal rye biomass reduced A. tuberculatus height, biomass, and density. An average ED50 of 5.2 Mg ha-1 of biomass was needed to reduce A. tuberculatus density by 50%. Low levels of biomass (≤ 2.38 Mg ha-1) augmented A. tuberculatus density due to an increase in soil moisture underneath the mulch compared to bare soil. Cereal rye biomass decreased the amount of sunlight reaching the soil, which resulted in lower mean soil temperature and temperature amplitude throughout the day (9.3 and 2.7 C temperature amplitude at 0 and 12.0 Mg ha-1, respectively). This thermal effect is likely the main mechanism of A. tuberculatus suppression from the cereal rye cover crop by not triggering A. tuberculatus germination. Our results support that biomass from cereal rye cover crop can effectively suppress A. tuberculatus and contribute to the integrated management of A. tuberculatus.