Intravenous fluids may contribute to lower haemoglobin levels in patients with septic shock. We sought to determine the relationship between the changes in haemoglobin concentration and the volume of intravenous fluids administered during resuscitation from septic shock. We performed a retrospective cohort study of patients enrolled in the Australasian Resuscitation in Sepsis Evaluation (ARISE) trial who were not transfused red blood cells (N = 1275). We determined the relationship between haemoglobin concentration, its change over time and volume of intravenous fluids administered over 6, 24 and 72 h using univariate and multivariate analysis. Median (IQR) haemoglobin concentration at baseline was 133 (118-146) g/L and decreased to 115 (102-127) g/L within the first 6 h of resuscitation (P < 0.001), 110 (99-122) g/L after 24 h, and 109 (97-121) g/L after 72 h. At the corresponding time points, the cumulative volume of intravenous fluid administered was 1.3 (0.7-2.2) L, 2.9 (1.8-4.3) L and 4.6 (2.7-7.1) L. Haemoglobin concentration and its change from baseline had an independent but weak association with intravenous fluid volume at each time point (R  < 20%, P < 0.001). After adjusting for covariates, each litre of intravenous fluid administered was associated with a change in haemoglobin concentration of - 1.0 g/L (95% CI -1.5 to -0.6, P < 0.001) at 24 h and - 1.3 g/L (- 1.6 to - 0.9, P < 0.001) at 72 h. Haemoglobin concentration decreases during resuscitation from septic shock, and has a significant but weak association with the volume of intravenous fluids administered.