The main pathophysiological mechanisms of hind limb ischemia–reperfusion injury (HLIRI) are increased oxidative stress and inflammation. The present study was designed to characterize the phytocontents of Dysphania ambrosioides (DA) seeds extract via LC-MS/MS and investigate its effect on left hindlimb IR injury and the underlying mechanisms. Thirty adult rats (n = 6 per group) were divided into five groups: Control group; HLIRI group, HLIRI + DA (100 mg/kg) group, HLIRI + DA (200 mg/kg) group, and HLIRI + cilostazol (30 mg/kg) group. HE staining’s of the left gastrocnemius muscle and left kidney was done. Renal function, and both total and muscle creatine kinases were measured in serum. Oxidative stress markers (MDA, SOD, Nrf2 and HO-1), inflammatory markers (NLRP3, IL-1β and TNF-α) and caspase-3 as an apoptotic marker were measured in left gastrocnemius muscle. We found that DA extract contains 60 metabolites and its pretreatment prevented left kidney and left gastrocnemius muscle damage and decreased oxidative stress markers, inflammatory markers, and caspase-3 levels, compared to the control group. Moreover, the effect of the extract was dose-dependent and better than that of the reference drug, cilostazol. This study suggested that D. ambrosioides seeds have a protective effect on HLIRI, which may be related to the antioxidant, anti-inflammatory and antiapoptotic mechanisms.