Botulinum neurotoxin A (BoNT/A), a 150 kDa di-chain protein produced by Clostridium botulinum, is used to treat chronic pain. It inhibits neuronal exocytosis by cleaving synaptosomal-associated protein 25 (SNAP-25) with its light chain protease (LC/A). The LC of serotype BoNT/E (LC/E) cleaves 17 more residues from SNAP-25 than LC/A; in vitro results have shown that unlike LC/A, the more extensive cleavage of SNAP-25 by LC/E prevents capsaicin-induced release of the pain mediating neuropeptide calcitonin gene-related peptide (CGRP). By attaching LC/E to BoNT/A, the short half-life of LC/E is extended to match BoNT/A, resulting in a chimera with potentially greater antinociceptive capabilities than BoNT/A. In the rat spared nerve injury (SNI) model of neuropathic pain, injections of LC/E-BoNT/A showed greater alleviation of mechanical pain than BoNT/A or LC/EBoTIM/A, a BoNT/A and LC/E chimera with an inactive LC/A. Unfortunately, intra-articular knee injections of LC/E-BoNT/A or BoNT/A induced confounding muscle paralysis, even at doses tolerated for intra-plantar injections; therefore, it was not feasible to inject enough neurotoxin to relieve symptoms of an inflammatory knee pain model. Injections of LC/E-BoNT/A prior to induction of nocifensive behaviours in the capsaicin or formalin models, resulted in more striking antinociceptive effects than BoNT/A pre-injections. LC/E-BoNT/A reduced formalin-induced phase I flinching behaviour and was comparable to sub-cutaneous buprenorphine treatment in relieving capsaicin-induced lifting/licking behaviours and heat hypersensitivity. However, these observations were inconsistent between batches of BoNTs produced; results from an in vitro substrate cleavage assay and in vivo dose escalation studies indicated activity loss of LC/E-BoNT/A over long-term storage, explaining some of the inconsistencies in anti-nociceptive effects. A fresh batch of LC/EBoNT/A demonstrated anti-nociceptive effects in the capsaicin and SNI model, but further symptoms of systemic toxicity than the previous batch.