Protein kinase C (PKC) and substrates like SNAP-25 regulate neurotransmission. At the neuromuscular junction (NMJ), PKC promotes neurotransmitter release during synaptic activity. Thirty minutes of muscle contraction enhances presynaptic PKC isoform levels, specifically cPKCβI and nPKCε, through retrograde BDNF/TrkB signaling. This establishes a larger pool of these PKC isoforms ready to promote neuromuscular transmission. The PKC phosphorylation site in SNAP-25 has been mapped to the serine 187 (Ser-187), which is known to enhance calcium-dependent neurotransmitter release in vitro. Here, we localize SNAP-25 at the NMJ and investigate whether cPKCβI and/or nPKCε regulate SNAP-25 phosphorylation. We also investigate whether nerve and muscle cell activities regulate differently SNAP-25 phosphorylation and the involvement of BDNF/TrkB signaling. Our results demonstrate that nPKCε isoform is essential to positively regulate SNAP-25 phosphorylation on Ser-187 and that muscle contraction prevents it. TrkB and cPKCβI do not regulate SNAP-25 protein level or its phosphorylation during neuromuscular activity. The results provide evidence that nerve terminals need both pre- and postsynaptic activities to modulate SNAP-25 phosphorylation and ensure an accurate neurotransmission process.