Two α-isoforms of the Na,K-ATPase (α and α) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of contractility. Mice heterozygous for the familial hemiplegic migraine type 2 (FHM2) associated mutation in the α-isoform (G301R; α mice) have decreased expression of cardiac α-isoform but elevated expression of the α-isoform. We aimed to investigate the contribution of the α-isoform function to the cardiac phenotype of α hearts. We hypothesized that α hearts exhibit greater contractility due to reduced expression of cardiac α-isoform. Variables for contractility and relaxation of isolated hearts were assessed in the Langendorff system without and in the presence of ouabain (1 µM). Atrial pacing was performed to investigate rate-dependent changes. The α hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α hearts, which was associated with increased systolic work.