Systemic inflammatory response syndrome (SIRS) is characterized by the activation of the innate immune system resulting in stimulation of inflammatory responses, coagulation, and platelet activation that may contribute to complication such as the development of acute kidney injury (AKI). AKI importantly worsens the outcome of SIRS, implying the existence of a detrimental cross talk via systemic messages. Mitochondria are a source of damage-associated molecular patterns (DAMPs) and are thought to form a molecular link between tissue injury and stimulation of innate immunity. The role of mitochondrial DNA (mtDNA) in the cross talk between the onset of SIRS and subsequent development of AKI is unknown. Hence, we performed a case control study in critically ill patients with SIRS diagnosed with or without AKI, in which we determined mtDNA levels in plasma and urine, and correlated these to markers of renal impairment, inflammation, coagulation, and platelet activation. In addition, we exposed mice, primary renal tubular epithelial cells (TECs), and platelets to mtDNA or purified mitochondrial ligands, and measured their response to elucidate underlying pathophysiological mechanisms. Our data reveal that increased systemic mtDNA levels in SIRS patients do not correlate with systemic inflammation and renal disease activity. Moreover, AKI does not have an additional effect on circulating mtDNA levels. In contrast, we found that urinary mtDNA levels correlate with an elevated albumin creatinine ratio (ACR) as well as with increased urinary markers of inflammation, coagulation, and platelet activation. Both renal TECs and platelets respond to mtDNA and mtDNA ligands, leading to increased expression of, respectively, inflammatory cytokines and P-selectin. Moreover, activation of platelets results in mtDNA release. Together, these data suggest that circulating mtDNA is probably not important in the detrimental cross talk between SIRS and AKI, whereas renal mtDNA accumulation may be related to intrarenal inflammation, coagulation processes, and renal dysfunction in the pathophysiology of SIRS.