The mammalian adenosine monophosphate-activated protein kinase (AMPK) is a serine-threonine kinase protein complex that is a central regulator of cellular energy homeostasis. However, the mechanisms by which AMPK mediates cellular responses to metabolic stress remain unclear. We found that AMPK activates transcription through direct association with chromatin and phosphorylation of histone H2B at serine 36. AMPK recruitment and H2B Ser(36) phosphorylation colocalized within genes activated by AMPK-dependent pathways, both in promoters and in transcribed regions. Ectopic expression of H2B in which Ser(36) was substituted by alanine reduced transcription and RNA polymerase II association to AMPK-dependent genes, and lowered cell survival in response to stress. Our results place AMPK-dependent H2B Ser(36) phosphorylation in a direct transcriptional and chromatin regulatory pathway leading to cellular adaptation to stress.