Background Class II a histone deacetylase ( HDAC ) isoforms such as HDAC 5 are critical signal‐responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor‐2. β‐Adrenoceptor (β‐ AR ) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation‐independent nuclear export and phosphorylation‐dependent nuclear accumulation of cardiomyocyte HDAC 5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC 5 regulation is also controversial. We aimed to determine the impact of β‐ AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC 5 and delineate underlying molecular mechanisms. Methods and Results A novel 3‐dimensional confocal microscopy method that objectively quantifies the whole‐cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC 5 revealed the β‐ AR agonist isoproterenol to induce β ₁ ‐ AR ‐mediated and protein kinase A‐dependent HDAC 5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC 5 nuclear accumulation and myocyte enhancer factor‐2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol‐induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP 2A blocked isoproterenol‐induced HDAC 5 dephosphorylation. Co‐immunoprecipitation revealed a specific interaction of HDAC 5 with the PP 2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3‐fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol‐induced HDAC 5 dephosphorylation. Conclusions β‐ AR stimulation induces HDAC 5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A‐dependent but requires B55α‐ PP 2A‐mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A‐mediated phosphorylation of Ser279.