Fruit acidity significantly influences fruit flavor, but the specific impact of soil pH on fruit acidity remains unclear. This study investigated the effects of various soil pH levels on fruit acidity and citric acid (CA) metabolism in lemon (Citrus limon L.). High soil pH (pH 8) decreased total soluble solids concentrations in lemon fruits, while low soil pH (pH 4) increased titratable acid and CA concentrations. Although low soil pH reduced the synthesis of CA due to the decreased citrate synthase and phosphoenolpyruvate carboxylase activities, the elevated fruit acidity under low soil pH conditions is not directly related to CA synthesis. Instead, low soil pH was found to suppress the activity of cytosolic aconitase (Cyt-ACO), an iron-dependent enzyme, indicating a potential role for CA degradation inhibition in low soil pH-induced CA accumulation. Furthermore, low soil pH significantly reduced cytosolic iron (Cyt-Fe) concentration, which was positively correlated with Cyt-ACO activity. In conclusion, low soil pH contributes to increasing fruit acidity in lemon, partially by inhibiting CA degradation due to the reduced Cyt-Fe concentrations. Our work unravels the influence of soil pH on CA accumulation and provides important clues for modulating CA levels through microelement fertilization in citrus.