The use of irrigation water containing arsenic (As) had led to large areas of As-contaminated farmland, and as a result, plants and food have become severely poisoned. Humic acid (HA) can be complexed with metals, which in turn affects the metals' behavior. Herein, we explored the accumulation of arsenate in lettuce treated with different concentrations of arsenate and studied the effects of HA on the accumulation and toxicity of arsenate. The addition of HA did not cause significant changes in the arsenate content in lettuce but had a significant effect on the activity of antioxidant enzymes, which improved the antioxidant capability of the lettuce plants. Furthermore, HA promoted the accumulation of nutrients, such as magnesium (Mg), calcium (Ca), molybdenum (Mo) and manganese (Mn), in the leaves. Arsenate disrupted metabolic pathways, such as amino acid metabolism, carbohydrate metabolism, and aminoacyl-tRNA biosynthesis. The addition of HA increased the contents of amino acids and sugars, thereby improving lettuce growth. The present study explored the effects of HA on As accumulation and related physiological changes (antioxidant enzyme activities, absorption of nutrients and metabolic mechanisms) and provided insights into the regulation of As contamination by HA, which is relatively inexpensive.