The sugar alcohol mannitol is an important carbohydrate in many plants andfungi that has well-documented roles in both metabolism and osmoprotection. In addition,mannitol is an antioxidant, and as such might play a role in host-pathogen interactions.Research suggests that pathogenic fungi secrete mannitol into the plant cell wall tosuppress reactive oxygen-mediated host defenses. Previous work suggests that plantscounter this by making the enzyme mannitol dehydrogenase (MTD) to catabolize fungalmannitol. Here we show that the normally cytoplasmic enzyme MTD is exported into theextracellular space in response to the endogenous inducer of plant defense responsessalicylic acid (SA). This SA-induced secretion is resistant to brefeldin A, an inhibitor ofGolgi-mediated protein transport. Together with the absence of MTD in Golgi stacks andthe lack of a documented extracellular targeting sequence in MTD, this suggests thesecretion of MTD is by a non-Golgi, pathogen-activated protein secretion mechanism inplants.To further characterize pathogen-activated protein secretion in plants, acomprehensive analysis was performed using Arabidopsis suspension culture to studytemporal changes in the cell wall proteome in response to different levels of SA. AnLC/MSE -based proteomic approach was used as a label-free approach for simultaneousprotein identification and absolute quantification. A total of 76 secreted proteins wereidentified, 66 of which showed differential secretion patterns in response to SA. Amajority of induced protein secretion was observed within the first two hours aftertreatment, suggesting many proteins are involved in the early stage of plant defenseresponse. A number of non-classically secreted proteins were observed, indicating that asin many non-plant systems, alternative Golgi/ER-independent secretion mechanismsmight exist in plants. Overall, our results provide new and useful insight into plantapoplastic defense mechanisms, and demonstrate that LC/MSE is a suitable strategy forabsolute quantitative proteomic analysis that can be applied for complex experimentaldesigns.