In the heart, nitric oxide (NO) modulates contractile function; however, the mechanisms responsible for this effect are incompletely understood. NO can elicit effects via a variety of mechanisms including S-nitrosylation and stimulation of cGMP synthesis by soluble guanylate cyclase (sGC). sGC is a heterodimer comprised of a β₁- and an α₁- or α₂-subunit. sGCα₁β₁ is the predominant isoform in the heart. To characterize the role of sGC in the regulation of cardiac contractile function by NO, we compared left ventricular cardiac myocytes (CM) isolated from adult mice deficient in the sGC α₁-subunit (sGCα₁^−/−) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα₁^−/− CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β₃-adrenergic receptor (β₃-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα₁^−/− myocytes. Administration of spermine NONOate, an NO donor compound, did not affect sarcomeric shortening in CM of either genotype; however, in the presence of isoproterenol, addition of spermine NONOate reduced sarcomere shortening in WT but not in sGCα₁^−/− CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα₁ exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β₃-AR signaling. Additionally, our work demonstrates that sGCα₁β₁ is required for NO to depress β₁/β₂-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.