Acetyl hexapeptide-3, a competitive SNAP-25 inhibitor that mimics the action of botulinum neurotoxins but is nontoxic, was successively synthesized in a solution by peptide chemistry with a "3+2+1" fragment coupling strategy. The difficulties encountered in the convergent route, an optimization on scalable process, and controls on potential epimerization are systematically discussed. Final purification led to the target product's overall 36.5% yield with 98.3% purity and excellent optical purity.