Extracorporeal membrane oxygenation (ECMO) is commonly used for critically ill patients and associated with high rates of kidney injury and mortality. This study aimed to assess the impact of diminished pulsatile flow induced by veno-arterial ECMO (V-A ECMO) on kidney structure and function. Ten healthy sheep were divided into two groups (n=5 each) to compare diminished pulsatile flow induced by V-A ECMO with normal pulsatile flow under veno-venous ECMO (V-V ECMO) over 7 days. Vital signs were recorded every 6 hours, and renal biomarkers were tested every 24 hours. The pulse pressure (PP) in the V-A ECMO group was around 20 mmHg, compared to 30 mmHg in the V-V ECMO group. The pulsatility index (PI) was lower than 0.25 in the V-A ECMO group and higher in the V-V ECMO group. Mixed model results showed significant differences in PP and PI between the two groups (p = 0.044 and p = 0.016, respectively). BUN and Cr levels were similar in both groups, but Cystatin C was higher in the diminished pulsatile flow group. The expression of anti-platelet endothelial cell adhesion molecule-1 (CD31) and the renin-positive glomerulus ratio were significantly lower in the diminished pulsatile flow group. Both groups exhibited hypoxia-inducible factor-1α (HIF-1α) in renal tubules, indicating hypoxia. These findings suggest diminished pulsatile flow under V-A ECMO affects renal endothelial cell proteins and renin secretion. During the management of ECMO, although the serum BUN and Cr were normal, there was exactly an early-stage injury of the glomerulus and tubules in both groups.