Abstract: In order to study the effects of low temperature stress on proline accumulation and the activities of the key enzymes involved in the proline metabolism, explore the relationship between proline metabolism and cold resistance, the experiment focused on determination of free proline content, pyrroline-5-carboxylate synthetase (P5CS) activity, ornithine-oxo-acid transaminase (8-0AT) activity, proline dehydrogenase (ProDH) activity, and analysis of dynamic changes of these indexes. Three tomato varieties, ‘Naiyun 2000’, ‘Jingle 502’, ‘O-33-1’, were used as the experimental materials. The results showed that the free proline accumulation increased and then decreased under low temperature stress, and the average proline content were lower in cold-sensitive ‘Naiyun 2000’, which can reflected the strength of cold resistance of tomato. Along with the increasing time of stress, the changes of P5CS activity and 8-0AT activity showed just same as the tendency of free proline. The ProDH activity increased obviously after one day while the free proline content decreased, and then showed a trend of decreased at first and then decreased which was contrary to the accumulation of free proline.