Highly pathogenic avian influenza H5N1 has been enzootic in Egypt since 2008. Virus-associated mortality (but not the number of cases) in humans and poultry seems to have decreased over time, but the reason for this remains unknown. We investigated the role of a single amino acid substitution in the hemagglutinin cleavage site on virus pathogenicity and transmission in chickens. The R325G substitution significantly reduced pathogenicity without altering the transmission efficiency of HPAI H5N1 virus.