Background: Shortages of allogeneic blood supplies for rhesus (Rh)-negative patients who are scheduled for major cancer surgeries may cause delays in surgical procedure, resulting in a prolonged length of hospital stay (LOHS). This study investigated the relationship of acute normovolemic hemodilution (ANH) with LOHS in this patient population.Methods: Rh-negative patients who underwent major cancer surgeries between January 2015 and April 2020 were included in this retrospective study. The primary outcome was LOHS. The secondary outcomes were length of preoperative stay (LOPS), perioperative laboratory data and allogeneic blood transfusion (ABT), and postoperative adverse events. Furthermore, relationships between these perioperative variables and LOHS were examined by both univariate analyses and multiple linear regression analysis.Results: Seventy patients were divided into ANH (n=30) or Control (n=40) group. The two groups were well-matched for baseline data. LOHS, LOPS, perioperative ABT amount, and the overall rate of postoperative adverse events were all significantly lower in the ANH group (P=0.004, P=0.009, P<0.001, P=0.023, respectively). In the ANH group, levels of hemoglobin and hematocrit decreased on postoperative day 1 (P=0.023, P=0.012, respectively). Univariate analyses revealed significant association between LOHS and the following perioperative variables: ANH, body mass index, types of surgery, intraoperative colloids infusion, and perioperative ABT. Multiple linear regression analysis with correction for diagnosis identified ANH, intraoperative colloids infusion, and perioperative ABT as independent predictors.Conclusions: ANH was associated with the decreased LOHS in Rh-negative patients undergoing major cancer surgeries.