Abstract Ample evidence suggests that infection with cytomegalovirus (CMV) leads to accelerated aging of the immune system and may contribute to poor responsiveness to influenza vaccination in older persons. The objective of this study was to investigate whether CMV infection, acquired earlier in life, affects the response to influenza vaccination in a randomized controlled trial among older persons in long-term care facilities. During the 1997–1998 influenza season, 731 residents (median age 83 [interquartile range 78–88], 75.4% female) in 14 long-term care facilities in the Netherlands were randomly assigned to receive 15 or 30μg of inactivated influenza vaccine, followed by a 15μg booster vaccine or a placebo vaccine at day 84. Blood samples were collected at day 0, day 25, day 84 and day 109. Seroresponses to influenza vaccination were measured by hemagglutination-inhibition tests to the A/H3N2 strain at all time points. Subsequently, baseline levels of IgG anti-CMV antibodies were measured using an automated chemiluminescent microparticle immunoassay. Participants with CMV antibody level ≥6AU/mL were considered to harbor CMV infection. At baseline, no differences in pre-vaccination geometric mean antibody titers (GMT) were observed between participants with (n=571, 78.1%) or without CMV infection (n=160, 21.9%). During follow-up, participants with and without CMV infection had similar responses to influenza vaccination as measured with changes in GMT (linear mixed model, adjusted for gender, age, pre-vaccination GMT and vaccination strategy, p=0.46). Analogously, no association was found between CMV infection and a more than 4-fold increase in antibody titer (Generalized Estimating Equations, adjusted OR 1.14 [95%CI 0.80;1.64]) or an antibody titer ≥40 (adjusted OR 1.24 [95%CI 0.86;1.80]). In conclusion, CMV infection did not explain poor responsiveness to influenza vaccination in residents of long-term care facilities.