AIMS: Heart failure with preserved ejection fraction (HFpEF) is an increasingly prevalent cardiovascular disease, which is often accompanied by kidney dysfunction. Exercise has been recognized as a feasible strategy to improve renal function. The aim of this study was to investigate whether aerobic exercise (AE) could ameliorate HFpEF-induced renal injury by regulating the expression of acyl-coenzyme A: lysocardiolipin acyltransferase-1 (ALCAT1). MATERIALS AND METHODS: Eight-week-old C57BL/6 and Alcat1 knockout mice were used to establish a HFpEF induced kidney injury model. Mice in the exercise-intervention group were performed a six-week of AE training. Cardiac function and blood pressure were assessed using echocardiography and a non-invasive intelligent blood pressure monitor. Renal morphology and function were detected by HE, Masson, and PAS staining, as well as biochemical assays using commercial kits. Oxidative stress, inflammation, apoptosis, and renal fibrosis-related proteins were detected by Western Blotting. KEY FINDINGS: In the HFpEF induced kidney injury model, ALCAT1 protein expression was upregulated, accompanied by cardiac and renal dysfunction. These pathological changes were reversed by AE. In addition, Alcat1 knockout significantly alleviated HFpEF-induced oxidative stress, inflammation, apoptosis, and fibrosis in the kidneys. Furthermore, Alcat1 knockout further enhanced the protective effects of exercise, ameliorating renal injury and improving renal function in HFpEF mice. SIGNIFICANCE: AE significantly improved renal function by alleviating oxidative stress, inflammation, apoptosis, and fibrosis in HFpEF mice. These beneficial effects were further enhanced in the lack of ALCAT1. Thus, ALCAT1 might represent a potential therapeutic target for the treatment of HFpEF-induced kidney injury.