Based on the known oxidative stress of nicotine, we examined the effects of Ucn I on nicotine-induced oxidative stress in HL-1 cardiomyocytes. HL-1 cardiomyocytes is plated on 96-well plate at a density of 2.0×104 cells/well with Claycomb medium containing 10% fetal bovine serum (FBS) and 0.1 mmol/L epinephrine. After starvation of FBS and epinephrine with Claycomb medium, cells were stimulated with or without (+/−)-nicotine, Ucn I and Ucn II. Cells were also stimulated with (+/−)-nicotine after knocking down of Ucn I mRNA. Oxidative stress is evaluated by conversion of 2′, 7′-dichlorodihydrofluorescin diacetate to 2′, 7′-dichlorodihydrofluorescein. (+/−)-nicotine exereted significant oxidative stress on HL-1 cardiomyocytes, while (−)-nicotine, a cis-trans isomer of (+/−)-nicotine, did not exert significant oxidative actions on HL-1 cardiomyocytes. Ucn I suppressed not only (+/−)-nicotine-induced oxidative stress, but also oxidative stress in standard culture condition in HL-1ardiomyocytes. In addition, knockdown of Ucn I by siRNA enhanced (+/−)-nicotine-induced oxidative stress. On the contrary, Ucn II alone, another agonist of corticotropin-releasing factor type 2 receptor, did not exert significant anti-oxidative stress action at the basal condition, but reduced (+/−)-nicotine-induced oxidative stress. The present results indicate that Ucn I may exert anti-oxidative actions on cardiomyocytes, of which mechanism of actions are still remained to be clarified.