Key points Epac (exchange protein directly activated by cyclic AMP (cAMP)), a PKA‐independent cAMP sensor, plays important roles in multiple cellular processes, but its role in the pathogenesis of skeletal muscle hypertrophy and myosin heavy chain (MHC) transition is poorly understood. Chronic stimulation of β2‐adrenoceptor (β2‐AR) with clenbuterol (CB), a selective β2‐AR agonist, induced masseter muscle hypertrophy in wild‐type (WT) mice, but not in Epac1‐null mice (Epac1KO), even if slow‐to‐fast MHC isoform transition was similarly induced by CB treatment in both WT and Epac1KO. Disruption of Epac1 inhibited development of masseter muscle hypertrophy concomitantly with decreased phosphorylation of Akt and its downstream molecules 70 kDa ribosomal S6 kinase 1 and eukaryotic initiation factor 4E‐binding protein 1, and also, in parallel, glycogen synthase kinase‐3β. Disruption of Epac1 decreased histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy. We conclude that Epac1 induces β2‐AR‐mediated masseter muscle hypertrophy without influencing slow‐to‐fast MHC isoform transition, probably via activation of Akt and its downstream molecules and increase of CaMKII‐mediated HDAC4 phosphorylation. AbstractThe predominant isoform of β‐adrenoceptor (β‐AR) in skeletal muscle is β2‐AR and that in the cardiac muscle is β1‐AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A‐independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2‐AR stimulation with clenbuterol (CB) in Epac1‐null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild‐type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB‐induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB‐mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB‐induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2‐AR‐mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.