Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However， it has lower binding affinity toward the human version of its major receptor， synaptotagmin II (h-Syt II)， compared to mouse Syt II， because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II， demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.Humans are less sensitive to the therapeutic effects of botulinum neurotoxin B (BoNT/B) than the animal models it is tested on due to differences between the human and the mouse receptors. Here， the authors engineer BoNT/B to improve its affinity to human receptors and enhance its therapeutic efficacy.