OBJECTIVE To evaluate the effect of healthcare worker (HCW) influenza vaccination on the incidence of nosocomial influenza DESIGN Retrospective cross-sectional study SETTING A 550-bed tertiary-care academic medical center METHODS All admitted patients with a direct fluorescent antibody (DFA) or polymerase chain reaction (PCR) assay positive for influenza ordered between October 1 and May 31 from 2010 to 2015 were eligible for inclusion. Nosocomial influenza was defined as a positive influenza test collected ≥48 hours after admission in patients without influenza-like illness present within 24 hours of admission. Relative nosocomial influenza frequency was calculated by dividing the number of nosocomial cases by the total number of admitted patients with influenza for each season. A univariate logistic regression was used to determine the association between HCW influenza vaccination coverage and nosocomial influenza. RESULTS Over 5 seasons, 533 patients had positive influenza tests during their hospitalization; 29 of these patients (5.4%) acquired influenza during their hospitalization. HCW vaccination coverage increased over the 5 seasons from 47% to 90% (P<.001). Despite an initial decrease in relative nosocomial influenza frequency during the first year (9% to 4.9%), subsequent seasons failed to show an additional decrease in nosocomial infections (4.3%, 5.2%, and 4.8%, respectively); the overall decrease in nosocomial influenza from the first season to the final season was not significant (P=.282). No association was detected between HCW vaccination coverage and nosocomial influenza (odds ratio [OR], 0.990; 95% confidence interval [CI], 0.970-1.011). CONCLUSION HCW vaccination >50% may not have a significant effect on nosocomial influenza. Infect Control Hosp Epidemiol 2016;37:840-844.