The difference between day and night temperature (DIF) is a major environmental factor affecting crop growth, but the mechanisms are not fully understood. We investigated crop performance, root activity and concentrations of low molecular weight organic acids (LMWOAs) secreted by tomato (Lycopersicon esculentum Mill.) root under different DIF conditions. A fixed daily temperature of 25°C and five DIF treatments (−12, −6, 0, 6 and 12°C) were used to grow tomato in a climate chamber. Root/shoot ratio; leaf maximum photosynthetic rate (P max); root activity; total nitrogen (N), phosphorus (P) and potassium (K) concentrations in roots; and types and concentrations of LMWOAs were measured at different growth stages. Results showed that positive and negative DIFs inhibited the dry matter accumulation of aerial parts, while 0°C DIF was conducive to the accumulation. Compared to 0°C DIF, positive DIFs significantly increased root dry weight, P max , root activity and total N, P and K concentrations in roots, while negative DIFs had contrary effects. During the whole growth period, tomato root activity decreased in the order of fruit setting stage, mature stage and flowering stage. Tomato roots secreted oxalic acid, formic acid, malic acid, malonic acid, lactic acid, acetic acid, citric acid, succinic acid and propionic acid under positive DIFs, while acetic acid was not detected in the negative DIF treatments. Oxalic acid concentration was significantly higher than other LMWOAs. Furthermore, in the same growth stage, positive DIFs caused more LMWOA secretion than negative DIFs and 0°C DIF. There were significant positive correlations between the total LMWOA concentration and root activity, root/shoot ratio, P max and total N, P and K concentrations in roots. Based on the results, more attention should be paid to the potential effect on tomato growth posed by DIFs, positive DIFs have higher positive influence than negative DIFs, and 6°C DIF is best for greenhouse tomato growth.