Plants involve a fine modulation of pectin methylesterase (PME) activity against microbes. PME activity can promote the cell wall stiffening and the production of damage signals able to induce defense responses and plant resistance to pathogens. However, the molecular mechanisms underlying PME activation during disease remain largely unknown. In this study, we explored the role of subtilases (SBTs) as PME activators in Arabidopsis immunity. By using biochemical and reverse genetic approaches, we found that the expression of SBT3.3 and SBT3.5 influences the induction of defense-related PME activity and resistance to the fungus Botrytis cinerea. Arabidopsis sbt3.3 and sbt3.5 knockout mutants showed decreased induction of PME activity and increased susceptibility to the fungus. SBT3.3 expression was stimulated by oligogalacturonides. Overexpression of SBT3.3 overactivated PME activity during fungal infection and enhanced resistance to B. cinerea. A negative correlation was observed between SBT3.3 expression and cell wall methyl ester content in the genotypes analyzed after B. cinerea infection. Increased expression of defense-related genes, including PAD3, CYP81F2 and WAK2, was also revealed in SBT3.3 overexpressing lines. We also demonstrated that SBT3.3 and pro-PME17 are both secreted into the cell wall using distinct protein secretion pathways and different kinetics. Our results propose SBT3.3 and SBT3.5 as modulators of PME activity in Arabidopsis against Botrytis to promptly boost immunity limiting the growth-defense trade-off.