Little is known about the association between intra-operative fluid balance (IFB) and post-operative acute kidney injury (AKI) in non-cardiac surgery. This is a retrospective cohort study. Adults who underwent non-cardiac surgery under general anesthesia from 2007 to 2011 at Nara Medical University Hospital were included. Those with obstetric or urological surgery, missing data, or pre-operative dialysis were excluded. Exposure of interest was IFB, defined as (amount of fluid administration - urine output - amount of bleeding)/body weight. Outcome variable was post-operative AKI within 1week after surgery. Data were analyzed using logistic regression models and restricted cubic spline (RCS) analysis. Among 5168 subjects, AKI was observed in 309 (6.0%). Higher IFB (per 1 standard deviation) was independently associated with post-operative AKI after adjustment for potential confounders (odds ratio [95% confidence interval] of 1.18 [1.06-1.31]). The RCS curve showed an increase in expected probability of AKI associated with increase in IFB above 40mL/kg. Subgroup analyses indicated higher IFB was especially associated with AKI among those with lower serum albumin, higher C-reactive protein, or positive proteinuria. The association was similar across intra-operative urine output or amount of bleeding (p for interaction 0.34 and 0.47, respectively), suggesting the association was not due to intra-operative oliguria or large amount of bleeding necessitating volume resuscitation. Higher IFB was independently associated with increase in post-operative AKI. Excessive fluid administration might have caused renal congestion and subsequent AKI. Avoiding fluid overload might be important in prevention of AKI.