We provide 2-D gel reference maps for the apoplastic proteome of Nicotiana benthamiana leaves infiltrated or not with the bacterial gene vector Agrobacterium tumefaciens. About 90 proteins were analyzed by LC-MS/MS for identification and function assignment. We show, overall, an effective response of the plant to agroinfiltration involving a specific, cell wall maintenance-independent up-regulation of defense protein secretion. The proteome maps described should be a useful tool for systemic studies on plant–pathogen interactions or cell wall metabolism. They also should prove useful for the monitoring of secreted recombinant proteins and their possible pleiotropic effects along the cell secretory pathway of N. benthamiana leaves used as an expression platform for clinically useful proteins.