Arsenic (As) is a widespread and highly toxic metalloid that adversely affects plant health, leading to reduced yields, impaired photosynthesis, and compromised root structures. Although acetylcholine (ACh), a neurotransmitter implicated in plant growth and stress responses, has shown potential in mitigating heavy metal stress, its effects on As stress in maize (Zea mays L.) remain largely unexplored. This study investigated the impact of ACh treatments on maize seedlings exposed to As stress (100 µM), focusing on physiological responses, growth rates, photosynthesis, and the antioxidant system. Our findings revealed that ACh treatments conferred differential protective effects against As stress. The lowest concentration (ACh1, 10 µM) enhanced growth by 2.6-fold compared to As-exposed plants, with no significant impact on relative water content (RWC). The medium concentration (ACh2, 50 µM) significantly improved both growth and RWC, effectively mitigating As-induced damage through enhanced osmotic adjustment and reduced oxidative stress. In contrast, the highest concentration (ACh3, 100 µM) yielded mixed results, showing improvements in RWC but minimal effects on growth, potentially due to metabolic imbalances from excessive ACh accumulation. Additionally, ACh2 treatment enhanced photosynthetic efficiency and increased the superoxide dismutase activity by 41% and catalase activity by 10%, facilitating the detoxification of radicals. By promoting the ascorbate–glutathione cycle, ACh supported redox homeostasis, demonstrating its protective role in alleviating As-induced oxidative damage and improving maize resilience under stress conditions.