Therapeutic use of botulinum neurotoxins is well established and is continuously expanding. In clinical practice, BoNT/A has been widely used to treat many pathological conditions characterized by autonomic overactivity. Current data suggest that BoNTs, given locally into peripheral tissues, alter nociceptive processing initiated by inflammation or nerve injury. This demonstration has recently allowed the use of BoNT/A as a novel treatment for a variety of pain syndromes, including neuropathic pain. However, it was observed that the use of BoNT/A over time can cause phenomena of immunogenicity and BoNT/B was proposed as a valid therapeutic alternative to BoNT/A. With the aim to assess the effect of BoNT/B on neuropathic pain, mice made neuropathic by Chronic Constriction Injury of sciatic nerve, were injected with a single dose of BoNT/B (5 or 7.5 pg) into the injured hind paw. The time course of the neuropathy was observed for 101 days and the effect of the neurotoxin on both pain and functional recovery was evaluated. For a comparison with functional data, immunofluorescence experiments in sciatic nerve and in spinal cord were associated to behavioral tests. The results of this study confirm BoNT/B as a powerful biological molecule that can reduce neuropathic pain for a long period of time. However, we provide the first evidence that BoNT/B also increases the expression of cells that release pro-inflammatory molecules in injured nerve and, in ventral horns of spinal cord, induces reactive astrogliosis development that could lead to glial scar formation and irreversibly compromise the functional recovery.