The hypoxic tumor microenvironment promotes both cancer progression and drug resistance.Prolyl hydroxylases are hypoxic sensor proteins that are frequently mutated or heterozygously deleted in breast cancers; however, how prolyl hydroxylation regulates breast cancer progression and drug resistance remains poorly understood. Here, we found that hypoxia promotes cancer progression and resistance to chemotherapy drugs, specifically through prolyl hydroxylation-dependent activation of MAPK signaling. We characterized a pVHL-independent mechanism wherein the prolyl hydroxylase EglN1 catalyzed prolyl hydroxylation of the proline-rich domain of RAS-specific guanine nucleotide exchange factor SOS1, thereby preventing its binding with the growth factor receptor bound protein 2 (GRB2) to block the formation of MAPK signaling microclusters in breast cancer. Inhibition of EglN1’s prolyl hydroxylase function—which occurs in hypoxic conditions and upon accumulation of fumarate—induced cancer-specific dysregulation of MAPK signaling, which promoted both breast cancer growth and resistance to MEK inhibitors. We harnessed this insight and demonstrated a therapeutic strategy that potently inhibits breast cancer growth and drug resistance based on pharmacologically disrupting the prolyl-hydroxylation-mediated interaction between SOS1 and GRB2.