Background and purpose: Increasing evidence suggests that the sympathetic nervous system profoundly interacts with skeletal muscle, influencing both muscle fiber function and composition. β2-Adrenoceptors (β2-ARs), the predominant adrenergic receptor subtype in muscle fibers, have been shown to enhance protein synthesis, reduce protein degradation, facilitate muscle contraction and relaxation, and improve neuromuscular junction (NMJ) transmission upon activation. In this study, we investigated the effects of Formoterol, a highly selective β2-AR agonist, on the presynaptic terminal of motor neurons. Experimental Approach: We used electromyography, FM1-43 fluorescent dye assays, and transmission electron microscopy to evaluate the neuromuscular junction following β2-receptor activation. Key Results: We demonstrated that β2-AR activation by Formoterol enhances muscle contractility and both spontaneous and evoked exocytosis of acetylcholine (ACh)-containing synaptic vesicles at the mouse diaphragm NMJ. Formoterol-induced morphological changes in diaphragmatic NMJs were consistent with increased exo-endocytic activity. Notably, Formoterol-evoked exocytosis displayed sexual dimorphism, with females showing a significantly milder response compared to males. In females, Formoterol-induced synaptic vesicles exocytosis was mediated solely by P/Q-type voltage-activated Ca2+ channels, whereas in males, it involved both P/Q-type channels, TRPV1 calcium channels, and an additional, yet unidentified, component. Orchiectomized males exhibited responses to Formoterol similar to the females., whereas ovariectomy did not modify female drug responses, indicating that male sex-hormonal environment orchestrates the sex-differences herein described. Conclusion and implications: These findings not only highlight the importance of sex-specific mechanisms but also reveal a novel effect of β2-AR activation directly on presynaptic terminals, enhancing exocytosis at the NMJ and thereby increasing neuromuscular transmission.