Freshly harvested tomato fruit (Solanum lycopersicum) were pre-treated with 0, 50, and 100 mM prohexadione-calcium (Pro-Ca) and then stored at 1oC for 21 days to investigate the effect of Pro-Ca treatment on electrolyte leakage (EL), malondialdehyde (MDA), proline and total phenols contents, and activity of phenylalanine ammonia-lyase (PAL) in relation to chilling injury (CI). Treatment with Pro-Ca, without significant difference between two applied concentrations, mitigated chilling injury, reduced EL and MDA content and increased proline content. Also, our results indicated that during storage time fruits treated with Pro-Ca exhibited significantly higher PAL activity, but total phenols content was not significantly affected by Pro-Ca treatments. These results suggested that Pro-Ca protects tomato fruit from CI by activation of PAL enzyme, enhancing proline contents and reducing MDA content, and thus maintaining membrane integrity.