Abstract Pro dehydrogenase (PDH) catalyzes the first and rate-limiting step in the Pro catabolic pathway. In Arabidopsis, this enzyme is encoded byAt-PDH. To investigate the role of Pro catabolism in plants, we generated transgenic Arabidopsis plants with altered levels of PDH by sense (PDH-S plants) and antisense (PDH-AS plants) strategies. Free Pro levels were reduced by up to 50% in PDH-S plants under stress and recovery conditions and enhanced by a maximum of 25% in PDH-AS plants, despite large modifications of theAt-PDH transcript and At-PDH protein levels. A similar trend in free Pro levels was observed in the PDH-S and PDH-AS seeds without visible effects on germination or growth. Under stress conditions, PDH transgenic plants showed no signs of change in osmotolerance. However, addition of exogenous Pro increased survival rates of salt-stressed PDH-S plants by 30%. Isotope-labeling studies showed that the conversion of [14C]Pro to Glu was reduced in PDH-AS plants and increased in PDH-S plants, especially under stress conditions. Furthermore, PDH-AS plants were hypersensitive to exogenous Pro, whereas PDH-S plants were sensitive to Pro analogs. These findings demonstrate that altered At-PDH levels lead to weakly modified free Pro accumulation with a limited impact on plant development and growth, suggesting a tight control of Pro homeostasis and/or gene redundancy.