Aim Atrial β2-adrenoceptors provide an important mechanism for regulation of cardiac function and changes in their downstream signaling are involved in processes underlying heart disorders. We have investigated the mechanism by which the cholesterol metabolite 5α-cholestan-3-one (5ɑCh3) modulates inotropic effect of β2-adrenoceptor agonist fenoterol. Main methods Atria from mice were electrically stimulated and changes in contraction amplitude in response to fenoterol were studied in 5ɑCh3-pretreated samples. Intracellular Ca^{2 +} and NO levels were estimated using fluorescent dyes Fluo-4 and DAF-FM, respectively. Key findings By itself 5αCh3 that appears in the circulation under some pathological conditions had a negligible influence on contraction, Ca^{2 +}-transient and NO production. However, pretreatment with 5αCh3 markedly attenuated the positive inotropic effect of fenoterol which was accompanied by an increase in the NO synthesis. Unexpectedly, the oxysterol also augmented an enhancement of Ca^{2 +}-transient amplitude in response to fenoterol. Under conditions of a pharmacological inhibition of G_i-protein/Akt/NO synthase/protein kinase G signaling, 5αCh3 augmented the inotropic effect of fenoterol. Herein, Akt antagonist suppressed the increase in NO production, while inhibition of NO synthesis did not modify the increased amplitude of the Ca^{2 +}-transient. Along similar lines, enrichment of plasma membranes with cholesterol reduced the stimulatory effect of 5αCh3 on β2-adrenoceptor-evoked NO production, but not on the Ca2 +-transient amplitude, leading to an elevation of the positive inotropic response to fenoterol. Significance These data suggest that 5ɑCh3 potentiates the effect of pharmacological β2-adrenoceptor activation on both NO production and Ca^{2 +} transient via independent mechanisms, thereby affecting the positive inotropy.