Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. Since the 1970s, many studies have focused on the role of GABA in the mammalian peripheral nervous system, and particularly in the cholinergic synapses. In this review, we present current findings for the cholinergic neurons of vegetative ganglia as well as for the neurons innervating smooth and striated muscles. Synaptic contacts formed by these neurons contain GABA and the enzyme, glutamic acid decarboxylase, which catalyzes the synthesis of GABA from glutamate. Newly formed GABA is released in the cholinergic synapses and mostly all the peripheral cholinergic synaptic contacts contain iono- and metabotropic GABA receptors. Although the underlying molecular mechanism of the release is not well understood, still, it is speculated that GABA is released by a vesicular and/or non-vesicular way via reversal of the GABA transporter. We also review the signaling role of GABA in the peripheral cholinergic synapses by modulating acetylcholine release, but its exact physiological function remains to be elucidated.