Background: Evidence regarding the effect of time to lactate measurement on the relationship between the initial lactate level and mortality is limited. We aimed to investigate the relationships between time to lactate measurement, initial lactate level, and in-hospital mortality in critically ill patients with sepsis.Methods and Results: Of the 14339 eligible adult patients with recognized sepsis upon admission to the ICU based on the MIMIC-III database, the median value of initial lactate was 1.70 mmol/L (interquartile range [IQR] 1.20-2.80), and its detection time was 3.50 hours ([IQR] 1.31-10.24). The results of fully adjusted multivariate analyses demonstrated that lactate was positively associated with in-hospital mortality (odds ratio: 1.126, 95% confidence interval: 1.090 to 1.163, P<0.001), and there was an increase in the odds of death with hourly delays in lactate measurement (OR: 1.006, 95% CI: 1.004 to 1.008, P<0.001). In stratified analyses, delays in lactate measurement significantly interfered with the impact of increased lactate level on mortality (P-value for interaction<0.001). The hospital mortality rate substantially increased by 43.5% for each unit increase in lactate when measurement was delayed by 24 hours (OR: 1.435, 95% CI: 1.260 to 1.635, P<0.001).Discussion: The association of initial lactate with in-hospital mortality is likely to vary with delays in detection time (grouping based on the “1-hour bundle”) in critically ill patients with recognized sepsis upon admission to the ICU.