Established quantitative standards for assessing decellularization of biologic scaffolds based on residual DNA levels have been well-documented and widely acknowledged. However, post-implantation complications, such as fever and seroma, are commonly observed which negatively impact clinical outcomes. The presence of cellular debris following decellularization or using source tissues that are naturally high in endotoxin may contribute to the host response to a biologic scaffold. In the study, several multi-step decellularization methods were used to decellularize small intestinal submucosa (SIS) to obtain materials with three distinct levels of residual DNA, lipid residues, and endogenous endotoxin. The potential influence of these residual components on macrophage and lymphocyte polarization in vitro, as well as on the host inflammatory response in vivo post intra-abdominal implantation or abdominal wall defect repair in rats, was assessed. Urinary bladder matrix (UBM) meeting established decellularization criteria and naturally devoid of endotoxin was utilized as a control. The presence of endogenous endotoxin in SIS-ECM resulted in notable changes in macrophage phenotype. SIS-ECM samples with endotoxin levels below FDA limits still upregulated pro-inflammatory factors in vitro. Conversely, SIS with minimal endotoxin content and UBM controls prompted a shift towards a pro-remodeling M2 phenotype, fostering constructive tissue remodeling in a rodent model of abdominal wall defects, irrespective of DNA content. These findings suggest that endotoxin may be a crucial factor influencing biologic scaffolds that are not fully accounted by current decellularization standards. STATEMENT OF SIGNIFICANCE: Clinically utilized decellularized biologic scaffolds that meet the established quantitative standards still suffer problems in high incidence of inflammatory complications, including fever and seroma. In this study, we confirmed that endotoxin, rather than residual DNA, is the crucial factor influencing host responses and regenerative outcomes. Tissue sources and decellularization processes are critical for reducing endotoxin levels and attenuating immuno-inflammatory complications. These findings enhance the evaluation of ECM scaffold performance for clinical application, thereby facilitating improved preparation and utilization for tissue defect repairs.