Fish are exposed to a wide variety of environmental stressors, such as chemicals and acute changes in temperature. Oxytetracycline (OTC) has been used as an antibiotic for many kinds of bacterial diseases in cultured fish, but excessive doses of OTC are known to cause side effects in fish and can have negative effects on their environment. In the present study, we examined stress-related biomarker expression in response to excessive doses of dietary OTC in coho salmon (Oncorhynchus kisutch). Fish received OTC (100 mg/kg body weight/day) orally for 2 weeks. The percentage of liver to body weight (hepatosomatic index; HSI) and plasma biochemical parameter, alanine aminotransferase (ALT) activity, of the group fed a diet containing OTC were observed to be significantly higher than those of the control group. The total glutathione (tGSH) levels in the liver of OTC-fed fish were four fold higher than those in control fish and double the control levels in muscle and stomach. Plasma tGSH levels in OTC-fed fish were also higher than those in control fish. Expression levels of heat shock protein 70 in the liver, muscle, and stomach decreased by OTC administration. Accordingly, OTC-induced stress might increase the metabolic turnover of GSH due to consumption by scavenging oxidants generated by stress. These results concerning the changing patterns of stress-related biomarkers indicate that excessive doses of OTC fed to coho salmon induce oxidative stress, which might enhance oxidation in the body and result in damage to tissues, especially in the liver. The present results also suggest that tissue-specific damage caused by OTC might already exist in fish.