As the incidence and length of spaceflight increases, the need to administer pharmacological agents to crew members may become greater. The final goal is to predict the therapeutic or toxic effects of drugs, especially those with a low therapeutic index, during spaceflight. The liver is central to systemic metabolism, therefore, various drugs rely on hepatic metabolism by cytochrome P450 (CYP). The function of individual CYP enzymes is diverse because they are subject to different regulatory mechanisms and substrate specificity. Because spaceflight may be a stressor and influence each CYP expression individually, the purpose of the present study was to measure the expression of 11 CYP genes and protein distribution in the liver of rats after spaceflight, using real-time polymerase chain reaction and immunohistochemistry, respectively. The gene and protein expression of stress-related proteins such as cold-inducible RNA binding protein (Cirp), heat shock protein 70 (HSP70), HSP90 and the p53 tumor suppressor gene, were also determined. CYP4A1and Cirp gene expression was significantly increased whereas HSP90 and p53 gene expression was significantly decreased in the flight group than in the ground control. Combined with histology, it is concluded that the effects of spaceflight on the liver may be similar to mild cold stress or fasting.