Recently, H9N2 avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Therefore, investigating the impact of its neuraminidase mutations on susceptibility to neuraminidase inhibitors (NAIs) is of great importance for clinically preventing potential cross-species transmission of H9N2. In this study, our results revealed that NAI resistance-related mutations of most H9N2 viruses isolated from China during 1998 to 2024 mainly occurred at positions N70S, E119A/D/G/V, Q133K, Q136A, D151E, Y155H, I222V, R224K, E276D, R292K, N294S, and R371K. Among them, the combined N70S and Y155H mutations are common characteristics of H9N2 viruses circulating in recent years. In vivo experiments demonstrated that these H9N2 viruses harboring the combined N70S and Y155H mutations exhibit high pathogenicity to BALB/c mice without prior adaption. In vitro neuraminidase inhibition assays confirmed that these H9N2 viruses bearing the N70S/Y155H mutations exhibited greater reduced inhibition than their counterparts bearing the N70S or Y155H single mutation to oseltamivir and zanamivir, but remained highly sensitive to peramivir. Notably, the in vivo protective effect of NAIs against H9N2 virus with combined N70S and Y155H mutations is limited, possibly due to the restricted ability of these drugs to ameliorate the excessive inflammatory responses. Therefore, there is an urgency to strengthen research on epidemiological surveillance and prevention strategies for avian influenza H9N2 harboring these mutations.