During flowering plant reproduction, anthers produce pollen grains, the development of which is supported by the tapetum, a nourishing maternal tissue that also contributes non-cell autonomously to the pollen wall, the resistant external layer on the pollen surface. How the anther restricts movement of the tapetum derived pollen wall components, whilst allowing metabolites such as sugars and amino acids to reach the developing pollen, remains enigmatic. Here we experimentally show that in Arabidopsis thaliana, the tapetum and developing pollen are symplastically isolated from each other, and from other sporophytic tissues, from meiosis onwards. We show that the peritapetal strip (PTS), an apoplastic structure, separates the tapetum and the pollen grains from other anther cell layers and can prevent the apoplastic diffusion of fluorescent proteins, again from meiosis onwards. The formation and selective barrier functions of the PTS require two NADPH oxidases, RBOHE and RBOHC, which play a key role in pollen formation. Together our results suggest that, together with symplastic isolation, gating of the apoplast around the tapetum may help generate metabolically distinct anther compartments.