A microneedle patch coated with vaccine simplifies vaccination by using a patch-based delivery method and targets vaccination to the skin for superior immunogenicity compared to intramuscular injection. Previous studies of microneedles have demonstrated effective vaccination using freshly prepared microneedles, but the issue of long-term vaccine stability has received only limited attention. Here, we studied the long-term stability of microneedles coated with whole inactivated influenza vaccine guided by the hypothesis that crystallization and phase separation of the microneedle coating matrix damages influenza vaccine coated onto microneedles. In vitro studies showed that the vaccine lost stability as measured by hemagglutination activity in proportion to the degree of coating matrix crystallization and phase separation. Transmission electron microscopy similarly showed damaged morphology of the inactivated virus vaccine associated with crystallization. In vivo assessment of immune response and protective efficacy in mice further showed reduced vaccine immunogenicity after influenza vaccination using microneedles with crystallized or phase-separated coatings. This work shows that crystallization and phase separation of the dried coating matrix are important factors affecting long-term stability of influenza vaccine-coated microneedles.