Accumulation of free L-proline (Pro) is a typical stress response incited by osmotic injuries in plants and microorganisms. Although the protective role of Pro in osmotic stress is not well understood, it is thought to function as compatible osmolyte or as a scavenger of reactive oxygen species (ROS). Here we show that, in Arabidopsis thaliana, Pro biosynthesis can be activated by incompatible plant-pathogen interactions triggering a hypersensitive response (HR). Pro accumulates in leaf tissues treated with Pseudomonas syringae pv. tomato avirulent strains (avrRpt2 and avrRpm1) but remains unchanged in leaves infected with isogenic virulent bacteria. Incompatible interactions lead to transcriptional activation of AtP5CS2, but not AtP5CS1, encoding the rate limiting enzyme in Pro biosynthesis pyrroline-5-carboxylate synthase (P5CS). AtP5CS2:GUS and AtP5CS2:LUC transgenes were induced inside and around the HR lesions produced by avirulent Pseudomonas spp. in transgenic plants. Pro accumulation was faster and stronger when stimulated by avrRpm1 than by avrRpt2, and was compromised in the low-salicylic acid plants NahG and eds5 when signaled through the RPS2-dependent pathway. In addition, Pro content and AtP5CS2 expression were enhanced by ROS in wild-type plants, suggesting that ROS may function as an intermediate signal in AtP5CS2-mediated Pro accumulation.