Abstract: Physiological responses to chilling, including antioxidative enzyme activity, Relative Water Content (RWC) and soluble sugar contents were investigated in soybean to identify mechanisms of chilling tolerance. Plants were exposed to 15°C (cold-acclimated) or 25°C (non-acclimated) for 24 h, under 250 μmol m-2 sec-1 Photosynthetically Active Radiations (PAR). Then all plants were exposed to 4°C (chilling temperature) for 24 h and allowed to recover at 25°C for 24 h. We analyzed the activity of ascorbate peroxidase (APX), catalase (CAT) and guaiacol peroxidase (GPX) and soluble sugar content and RWC in both shoots and roots of soybean seedlings. It revealed that the activity of APX and CAT and GPX induced in leaves and roots. Increased activity in roots is important for cold tolerance as compared to shoots. The amount of RWC decreased in both roots and shoots, but soluble sugar content increased, especially in shoots as compared to control plants. Chilling sensitive soybean plants can be made tolerant to cold by cold-acclimation.