Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H2O2 and the hydroxyl radical (•OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H2O2, •OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H, 23H-porphin, or a spin-trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, which eliminates •OH. The inhibiting concentrations of exogenous H2O2 and •OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo-osmotic shock. •OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H2O2, as well as partial removal of endogenous •OH, over-tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H2O2-inducible inter-polymer cross-linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross-links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. •OH is involved in local loosening of the intine in the germination pore region, while H2O2 is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.