γ-Aminobutyric acid (GABA) has a significant impact on the functioning of not only the central but also the peripheral part of the nervous system. Recently, various elements of the GABAergic signaling system have been discovered in the area of the neuromuscular junction of mammals. At the same time, the functional activity of membrane-bound GABA transporters (GATs) and their role in neuromuscular transmission have not been identified. In the present study, performed on a neuromuscular preparation of the mouse diaphragm, the effect of GABA transporter inhibitors (nipecotic acid and β-alanine) on the force of muscle contraction was assessed. It was found that in the presence of both compounds in the bathing solution, the force of contractions caused by stimulation of the motor nerve dropped by 30–50%. However, when the muscle was stimulated directly, no effect of GABA transporter inhibitors on the contractile force was observed. The depressant effect of β-alanine induced by nerve stimulation was completely abolished by the GABA_B receptor blocker CGP 55845. GABA transporters were detected at the neuromuscular junction using immunohistochemistry. Thus, our results indicate that GABA transporters are localized in the area of the neuromuscular junction, and their activity affects the muscle contraction force. This influence is most likely due to the removal of GABA released during nerve stimulation and activating GABA receptors, which leads to a decrease in the contraction force of the striated muscles.