Background: It remains unclear whether the mean vancomycin trough concentration (VTC) derived from the entire course of therapy is of potential benefit for critically ill patients. This study was conducted to explore the association between mean serum VTC and mortality in intensive care units (ICUs).Methods: 3,364 adult patients with two or more VTC records after receiving vancomycin therapy in the eICU Collaborative Research Database were included in this multicenter retrospective cohort study. Mean VTC was estimated using all measured VTCs and investigated as a continuous and categorical variable. Patients were categorized into four groups according to mean VTC: <10, 10–15, 15–20, and >20 mg/L. Multivariable logistic regression and subgroup analyses were performed to investigate the relationship of mean VTC with mortality.Results: After adjusting for a series of covariates, logistic regression analyses indicated that mean VTC, as a continuous variable, was positively correlated with ICU (odds ratio, 1.042, 95% confidence interval, [1.017–1.068]) and hospital (1.025 [1.004–1.046]) mortalities. As a categorical variable, mean VTC at 10–15 mg/L failed to reduce ICU mortality (1.512 [0.849–2.694]). Moreover, mean VTCs of 15–20 and >20 mg/L were significantly associated with higher ICU mortality (1.946 [1.106–3.424]; 2.314 [1.296–4.132]) than mean VTC <10 mg/L. Mean VTCs of 10–15, 15–20, and >20 mg/L were not associated with increased hospital mortality (1.154 [0.766–1.739]; 1.342 [0.896–2.011]; 1.496 [0.981–2.281]). Similar results were observed in different Acute Physiology and Chronic Health Evaluation IV score or creatinine clearance subgroups.Conclusions: Increasing mean VTC showed no benefit regarding ICU and hospital mortalities in critically ill patients. Our results suggested that continuous VTC monitoring might not guarantee vancomycin efficacy for ICU patients.