AbstractAnabolic β₂-adrenoceptor (β₂-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β₂-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β₂-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β₂-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks’ treatment with the β₂-AR agonist formoterol and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β₂-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β₂-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β₁- and β₂-ARs. These findings demonstrate that gene therapy-based interventions targeting the β₂-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration and highlight novel methods for potentially modulating muscle mass in settings of disease.