Real-time dissection of dynamic uncoating of individual influenza virusesInfluenza A virus (IAV) is one of the most important human pathogens, and it is crucial to understand its life cycle to develop antiviral strategies. However, IAV uncoating, an essential step in viral infection, has remained incomprehensible. Here, via the construction of infectious IAV virions encapsulating quantum dots, we tracked the uncoating and viral ribonucleoprotein complex (vRNP) dynamics of single IAV virions. Our results reveal that after viral fusion and uncoating, IAV vRNP segments are released separately into the cytosol, and individual vRNPs undergo a three-stage active nuclear import process and display two diffusion patterns within the nucleus. These findings reveal uncoating and vRNP trafficking mechanisms which may assist in developing new strategies to block IAV infection. Uncoating is an obligatory step in the virus life cycle that serves as an antiviral target. Unfortunately, it is challenging to study viral uncoating due to methodology limitations for detecting this transient and dynamic event. The uncoating of influenza A virus (IAV), which contains an unusual genome of eight segmented RNAs, is particularly poorly understood. Here, by encapsulating quantum dot (QD)-conjugated viral ribonucleoprotein complexes (vRNPs) within infectious IAV virions and applying single-particle imaging, we tracked the uncoating process of individual IAV virions. Approximately 30% of IAV particles were found to undergo uncoating through fusion with late endosomes in the “around-nucleus” region at 30 to 90 minutes postinfection. Inhibition of viral M2 proton channels and cellular endosome acidification prevented IAV uncoating. IAV vRNPs are released separately into the cytosol after virus uncoating. Then, individual vRNPs undergo a three-stage movement to the cell nucleus and display two diffusion patterns when inside the nucleus. These findings reveal IAV uncoating and vRNP trafficking mechanisms, filling a critical gap in knowledge about influenza viral infection.