Na,K-ATPase is composed of two essential α- and β-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its α 1, α 3 and β 1 isoforms are reduced in the failing human heart. The catalytic α-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase β 1-subunit (Na,K-β 1) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-β 1 gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-β 1 gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13–14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-β 1 KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-β 1 KO in older mice. Further, we show that intact hearts of Na,K-β 1 KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-β 1 plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.