Influenza A virus (IAV) is a major pathogen that threatens human and animal health. In June 2022, seven golden monkeys (Rhinopithecus roxellana) developed flu-like symptoms in succession at a zoo in Jiangsu Province, China, with two succumbing to respiratory distress. Histopathological and immunostaining results supported a diagnosis of pulmonary infection with IAV, and a novel H3N2 canine/human reassortant virus, designated A/golden monkey/Jiangsu/1/2022 (Gm-1), was isolated from the lungs of deceased animals. Genomic sequencing revealed that the PB1 gene of Gm-1 exhibited >?97?% identity with human strains, while the remaining 7 segments originated from early local canine H3N2 virus (A/canine/Jiangsu/06/2010, JS06). To determine whether the human-origin PB1 segment conferred a virulence advantage to Gm-1, we reconstructed this reassortant event using reverse genetics, generating two reassortment viruses, rGm-1 (human-origin PB1) and rJS06 (canine-origin control). Mice infected with rGm-1 showed more severe lung pathology and elevated proinflammatory cytokine levels compared to rJS06, indicating that the human-origin PB1 enhanced viral virulence. Further analysis identified a unique cytotoxic motif (68I, 69?L and 70?V) in PB1-F2 protein of Gm-1, absent in JS06, which may contribute to increased pathogenicity. This is the first report confirming IAV infection in golden monkeys. Our finding highlight the importance of enhanced biosecurity surveillance for this endangered species.