Subtype H3N2 influenza A viruses (IAVs), which emerged in 1968 to cause a pandemic, have shown continual circulation and adaptation that has necessitated frequent updates of candidate vaccine viruses. Here, we sought to determine how genetic changes in the hemagglutinin (HA) and neuraminidase of 21 antigenically distinct H3N2 IAVs isolated from 1968 to 2019 correlate with mammalian fitness and adaptation. We found a surge of adaptation between 1997 and 2002, resulting in the emergence of A/Fujian/411/2002 (H3N2) and poor vaccine efficacy, leading to an epidemic during the 2003-2004 season. This surge was characterized by a large reduction in binding to mammalian-type α2,6-linked sialic acids and increased infectivity and replication kinetics in humanized Madin-Darby canine kidney cells. HA glycosylation also increased most rapidly from 1968 to 2004 and then plateaued. Symptomatic infections were only evident in mice following inoculation with viruses isolated in the 1970s and A/Aichi/2/1968 (H3N2), which was the most pathogenic. More recent viruses did not cause any detectable symptoms, except for A/Sydney/5/1997 (H3N2), which caused some weight loss. The post-2002 shift to α2,6-linked sialic acid binding, coupled with reduced pathogenicity in mammalian models, underscores H3N2 adaptation to human circulation without affecting immunogenicity, which is a critical consideration for vaccine design. Overall, our data revealed that a surge of mammalian adaptation from 1997 to 2002 gave rise to A/Fujian/411/2002 (H3N2), with subsequent viruses showing more hallmarks of mammalian adaptation, such as increased binding to cells expressing α2,6-linked sialic acids and reduced mammalian pathogenicity.IMPORTANCEThe continued endemicity of subtype H3N2 influenza A viruses (IAVs) in humans necessitates an understanding of the continuing accumulation of mammalian adaptations to inform public health countermeasures. We used a combined approach of studying the genetic, antigenic, and pathogenic adaptations of the surface glycoproteins of 21 H3N2 IAVs representing distinct antigenic groups from 1968 to 2019. We observed a loss of mammalian pathogenicity within 10 years of human circulation and a surge of adaptation between 1997 and 2002 that gave rise to A/Fujian/411/2002 (H3N2), which was a poor match for vaccine viruses. This surge was characterized by large shifts in glycan binding preferences, antigenicity, and genetic evolutionary distances. Overall, our study reveals novel insights into the chronology of the mammalian adaptation of H3N2 IAVs.