Endurance exercise could attenuate obesity induced by high fat diet (HFD). Thus, the purpose of this study was to explore the crucial targets that play key roles in the improvement of body fat index (BFI) in obese mice by endurance exercise. Firstly, we constructed murine obesity models: High fat diet control (HFD) group, HFD exercise (HFE) group, normal chow diet control (NC) group, and normal chow diet exercise (NE) group. Next, we identified the BFI improvement related genes using differential gene analysis, and investigated these genes’ functional pathways using functional enrichment analysis. The qRT-PCR and western blot assays were used to determine the gene expression and protein expression, respectively. Gene set enrichment analysis was used to explore the potential pathways associated with endurance exercise in obese mice and Mitochondrial respiratory control ratio (RCR) assay was applied to determine the RCR in the liver tissues of mice. We discovered that endurance exercise remarkably reduced the body weights and BFI of HFD-induced obese mice. Runx1t1 was related to the improvement of BFI by endurance exercise in HFD-induced obese mice. Runx1t1 mRNA and protein levels in liver tissues were observably decreased in HFD mice compared to mice in HFE, NC and NE groups. Moreover, Glucagon signaling pathway that was associated with mitochondrial function was significantly activated in HFE mice. The Runx1t1 expression exhibited an observable negative correlation with Acaca in HFD mice. Moreover, the mitochondrial RCR level was significantly increased in HFE mice than that in HFD mice. In HFD-induced obese mice, Runx1t1 was implicated in the improvement of BFI via endurance exercise. Endurance exercise could improve mitochondrial dysfunction in obese mice by activating the Runx1t1.