Estimates of rotavirus vaccine effectiveness (VE) in the United States appear higher in years with more rotavirus activity. We hypothesized rotavirus VE is constant over time but appears to vary as a function of temporal variation in local rotavirus cases and/or misclassified diagnoses. We analyzed 6 years of data from eight US surveillance sites on 8- to 59-month olds with acute gastroenteritis symptoms. Children's stool samples were tested via enzyme immunoassay (EIA); rotavirus-positive results were confirmed with molecular testing at the US Centers for Disease Control and Prevention. We defined rotavirus gastroenteritis cases by either positive on-site EIA results alone or positive EIA with Centers for Disease Control and Prevention confirmation. For each case definition, we estimated VE against any rotavirus gastroenteritis, moderate-to-severe disease, and hospitalization using two mixed-effect regression models: the first including year plus a year-vaccination interaction, and the second including the annual percent of rotavirus-positive tests plus a percent positive-vaccination interaction. We used multiple overimputation to bias-adjust for misclassification of cases defined by positive EIA alone. Estimates of annual rotavirus VE against all outcomes fluctuated temporally, particularly when we defined cases by on-site EIA alone and used a year-vaccination interaction. Use of confirmatory testing to define cases reduced, but did not eliminate, fluctuations. Temporal fluctuations in VE estimates further attenuated when we used a percent positive-vaccination interaction. Fluctuations persisted until bias-adjustment for diagnostic misclassification. Both controlling for time-varying rotavirus activity and bias-adjusting for diagnostic misclassification are critical for estimating the most valid annual rotavirus VE.