Abstract Maize (Zea mays) kernels are the largest cereal grains, and their endosperm is severely oxygen deficient during grain fill. The causes, dynamics, and mechanisms of acclimation to hypoxia are minimally understood. Here, we demonstrate that hypoxia develops in the small, growing endosperm, but not the nucellus, and becomes the standard state, regardless of diverse structural and genetic perturbations in modern maize (B73, popcorn, sweet corn), mutants (sweet4c, glossy6, waxy), and non-domesticated wild relatives (teosintes and Tripsacum species). We also uncovered an interconnected void space at the chalazal pericarp, providing superior oxygen supply to the placental tissues and basal endosperm transfer layer. Modeling indicated a very high diffusion resistance inside the endosperm, which, together with internal oxygen consumption, could generate steep oxygen gradients at the endosperm surface. Manipulation of oxygen supply induced reciprocal shifts in gene expression implicated in controlling mitochondrial functions (23.6 kDa Heat-Shock Protein, Voltage-Dependent Anion Channel 2) and multiple signaling pathways (core hypoxia genes, cyclic nucleotide metabolism, ethylene synthesis). Metabolite profiling revealed oxygen-dependent shifts in mitochondrial pathways, ascorbate metabolism, starch synthesis, and auxin degradation. Long-term elevated oxygen supply enhanced the rate of kernel development. Altogether, evidence here supports a mechanistic framework for the establishment of and acclimation to hypoxia in the maize endosperm.