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Background While perioperative blood transfusion (BT) has been associated with adverse outcomes in multiple malignancies, the importance of BT timing has not been established. Objective The objective of this study was to evaluate whether intraoperative BT is associated with worse cancer outcomes in bladder cancer patients treated with radical cystectomy (RC). Design, setting, and participants Outcomes from two independent cohorts of consecutive patients with bladder cancer treated with RC were analyzed. Outcome measurements and statistical analysis Recurrence-free survival, cancer-specific survival (CSS), and overall survival were estimated and multivariate analyses were performed to evaluate the association of BT timing with cancer outcomes. Results and limitations In the primary cohort of 360 patients, 241 (67%) received perioperative BT, including 162 intraoperatively and 79 postoperatively. Five-year CSS was 44% among patients who received an intraoperative BT versus 64% for patients who received postoperative BT ( p = 0.0005). After multivariate analysis, intraoperative BT was associated with an increased risk of cancer mortality (hazard ratio [HR]: 1.93; p = 0.02), while receipt of postoperative BT was not ( p = 0.60). In the validation cohort of 1770 patients, 1100 (62%) received perioperative BT with a median postoperative follow-up of 11 yr (interquartile range: 8.0–15.7). Five-year RFS ( p < 0.001) and CSS ( p < 0.001) were significantly worse among patients who received an intraoperative BT. Intraoperative BT was independently associated with recurrence (HR: 1.45; p = 0.001), cancer-specific mortality (HR: 1.55; p = 0.0001), and all-cause mortality (HR: 1.40; p < 0.0001). Postoperative BT was not associated with risk of disease recurrence or cancer death. Conclusions Intraoperative BT is associated with increased risk of bladder cancer recurrence and mortality. Patient summary In this study, the effects of blood transfusion on bladder cancer surgery outcomes were evaluated. Intraoperative blood transfusion, but not postoperative transfusion, was associated with higher rates of recurrence and cancer-specific mortality. [ABSTRACT FROM AUTHOR]