Selenoprotein K (SelK) is a newly identified selenoprotein. We showed that selenium incorporation into SelK was dependent on the 3'UTR of SelK mRNA. Sec insertion sequence (SECIS) RNA binding assays demonstrated that human SBP2 bound to the SelK SECIS element through the conserved non-Watson-Crick base pair quartet but not the AAT motif. Examination of the expression pattern revealed that human SelK mRNA was highly expressed in heart. Immunofluorescence analysis showed that SelK localized to the endoplasmic reticulum. Using SelK recombinant adenovirus, we found that overexpression of SelK attenuated the intracellular reactive oxygen species level and protected cells from oxidative stress-induced toxicity in cardiomyocytes. Our findings indicated that SelK is a novel antioxidant in cardiomyocytes and is related to the regulation of cellular redox balance.