The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI)-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI) through epigenetic mechanisms. Interestingly, Cr(VI) exposure also results in the loss of acetylation at histone H4K16, which is considered a 'hallmark' of human cancer. Cr(VI)-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a) overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1), which specifically acetylates H4K16; (b) the loss of acetylation of H4K16 upon Cr(VI) exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI)-induced cell transformation. We propose that Cr(VI) induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI)-induced carcinogenesis.