The heart is one of the most frequently affected organs in sepsis. Recent studies focused on lipopolysaccharide-induced mitochondrial dysfunction; however myocardial dysfunction is not restricted to gram-negative bacterial sepsis. The purpose of this study was to investigate circulating heparan sulfate (HS) as an endogenous danger associated molecule causing cardiac mitochondrial dysfunction in sepsis. We used an in vitro model with native sera (SsP) and sera eliminated from HS (HS-free), both of septic shock patients, to stimulate murine cardiomyocytes. As determined by extracellular flux analyzing, SsP increased basal mitochondrial respiration, but reduced maximum mitochondrial respiration, compared with unstimulated cells (P < 0.0001 and P < 0.0001, respectively). Cells stimulated with HS-free serum revealed unaltered basal and maximum mitochondrial respiration, compared with unstimulated cells (P = 0.1174 and P = 0.8992, respectively). Cellular ATP-level were decreased in SsP-stimulated cells but unaltered in cells stimulated with HS-free serum compared with unstimulated cells (P < 0.0001 and P = 0.1593, respectively). Live-cell imaging revealed an increased production of mitochondrial reactive oxygen species in cells stimulated with SsP compared with cells stimulated with HS-free serum (P < 0.0001). Expression of peroxisome proliferator-activated receptors (PPARα and PPARγ) and their co-activators PGC-1α, which regulate mitochondrial function, were studied using PCR. Cells stimulated with SsP showed downregulated PPARs and PGC-1α mRNA-levels compared with HS-free serum (P = 0.0082, P = 0.0128, and P = 0.0185, respectively). Blocking Toll-like receptor 4 revealed an inhibition of HS-dependent downregulation of PPARs and PGC-1α (all P < 0.0001). In conclusion, circulating HS in serum of septic shock patients cause cardiac mitochondrial dysfunction, suggesting that HS may be targets of therapeutics in septic cardiomyopathy.