Butachlor is an herbicide extensively employed in rice cultivation, which has historically been under-investigated for its toxicological impacts on terrestrial vegetation. This study examines the dose-dependent effects of butachlor on the germination and antioxidant defense mechanisms in the seeds of Asian tape grass [Vallisneria natans (Lour.) H. Hara], an important submerged plant species widely distributed in the agricultural ponds. Employing a hydroponic setup, seeds were exposed to four concentrations of butachlor (0, 20, 200, and 2000 μg active ingredients L-1), and cultivated under controlled light conditions to quantify germination rates and assess oxidative stress responses. Our findings showed that butachlor concentrations up to 20 μg L-1 had no effect on the germination rate of V. natans seeds, while germination rates decreased by 6.0% and 8.7% at 200 and 2000 μg L-1, respectively. At 2000 μg L-1, malondialdehyde (MDA) content increased by 5.7 nmol g-1 FW, and catalase (CAT) activity declined by 21%, indicating oxidative damage. Additionally, the antioxidants proline (Pro) and glutathione (GSH) were upregulated under 20 μg L-1 butachlor treatment after 12 h, contributing to reactive oxygen species (ROS) scavenging and cellular stability. This study highlights the nuanced interactions between butachlor exposure and the antioxidant defenses in V. natans, providing valuable insights into the ecological impacts of herbicide pollution. Understanding these interactions is crucial for developing sustainable agricultural practices and for the management of herbicide resistance in aquatic systems.