Tobacco use significantly increases the risk of developing cancer. Moreover, there is growing evidence that tobacco use decreases survival in cancer patients. Nicotine, a systemically available component of tobacco, is associated with tumor promotion and decreased apoptosis in cell culture; however, the role of nicotine on response to radiotherapy (RT) or chemoradiotherapy (CRT) in vivo has not been evaluated. Our study evaluated the effects of nicotine administration on cancer cell survival in cell culture and mouse models. Nicotine increased survival in two cell lines following RT in vitro. Nicotine administration in mice during fractionated RT or CRT increased xenograft regrowth as compared to RT or CRT alone. Nicotine increased hypoxia-inducible factor 1-alpha (HIF-1α) expression in tumor xenografts without altering expression of carbonic-anhydrase, a clinical marker of tumor hypoxia. The effects of nicotine on HIF-1α expression were transient, returning to baseline levels within 2-3 days after nicotine removal. Further mechanistic studies indicated that inhibition of phosphoinositide-3-kinase (PI3K) prevented nicotine-mediated increases in HIF-1α expression as well as the prosurvival effects of nicotine on RT. These findings imply that during tobacco use, nicotine may function as a systemic agent through acute and reversible regulation of HIF-1α expression and a decreased therapeutic response.