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The sporadic outbreaks of highly pathogenic H5N1 avian influenza virus have raised public health concerns. Monoclonal antibodies (MAbs) against hemagglutinin (HA) have been increasingly used successfully for therapeutic purposes. Previously, MAb 9F4, generated against clade 1 H5N1 HA, was observed to have cross-clade neutralizing efficacy and inhibited viral entry by preventing the pH-mediated conformational change of HA. Furthermore, mouse-human chimeric MAb 9F4 was found to retain high degrees of neutralizing activity. In this study, through escape mutant generation and in-silico prediction, it was revealed that MAb 9F4 binds to a novel epitope in the vestigial esterase sub-domain of HA comprising at least three non-continuous amino acid residues, arginine (R) at position 62, tryptophan (W) at position 69 and phenylalanine (F) at position 79, which interacted with MAb 9F4 in a conformation-dependent manner. Binding and neutralization studies suggested that R62 is the critical residue for MAb 9F4 binding whereas W69 and F79 seem to cooperate with R62 to stabilize the epitope. Mutation of either R62 or W69 did not affect replicative fitness of the virus in vitro. Interestingly, MAb 9F4 retained neutralizing efficacy against a clade 220.127.116.11a H5N1 virus consisting of an arginine to lysine substitution at position 62 in HA.Monoclonal antibody based approaches against H5N1 may useful during pandemic situations.Murine monoclonal antibody 9F4 binds to a novel epitope in the vestigial esterase sub-domain of the hemagglutinin protein.Monoclonal antibody 9F4 retains neutralizing efficacy against a recent clade 18.104.22.168a H5N1 virus.Monoclonal antibody 9F4 may be further studied for its potential use in passive immunotherapy.