MRI using hyperpolarized 13C‐labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor‐bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO2) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1‐13C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast‐enhanced‐MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X‐irradiation was delivered during the period of pyruvate‐induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO2 after pyruvate injection in hyperpolarized 13C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.