Background/Objectives: The strategy of targeting endotoxins and circulating histones to alleviate excessive inflammation and tissue damage has been proposed as an important immunoregulatory strategy against sepsis. However, the development of a multifunctional hemoperfusion adsorber that simultaneously removes endotoxins and histones remains an unmet clinical need in sepsis management. Methods: We synthesized chitosan-cellulose composite (CSCE) microspheres utilizing phase inversion technology, while heparin-grafted chitosan-cellulose composite (CSCEHEP) microspheres were developed by grafting heparin onto CSCE microspheres through the carbodiimide coupling method. In our experimental design, we allocated healthy New Zealand rabbits into four distinct groups: a healthy control group, a lipopolysaccharides (LPS) group, a CSCE group, and a CSCEHEP group. Following the administration of LPS for 12 hours, septic rabbits underwent extracorporeal hemoperfusion with either CSCE or CSCEHEP microspheres for a duration of 2 hours, notably without the inclusion of heparin in the blood circuits. Post-hemoperfusion, we conducted an analysis of thrombus formation and total protein adsorption on the column. Concurrently, blood samples were collected from the venous side to evaluate LPS levels, histone presence, inflammatory cytokine concentrations, and to perform histopathological assessments of liver and kidney injury. Results: Our in vivo experiments demonstrated that CSCEHEP microspheres for extracorporeal circulation could achieve 2 hours hemoperfusion session in septic rabbits without the need for continuous anticoagulation with heparin. CSCEHEP column turn into a very light red color (almost original white) and no obvious contamination or clotting was observed after the 2 hours hemoperfusion. Moreover, CSCEHEP microspheres effectively reduced the concentration levels of leukocyte, serum IL-6 and TNF-α, mitigated pathological damage to the liver and kidneys, and removed over 70% of LPS and nearly 50% of histone H3 from the blood of septic rabbits during hemoperfusion. In conclusion, hemoperfusion utilizing CSCEHEP microspheres exhibits excellent self-anticoagulation capabilities, remarkable anti-inflammatory performance, efficient endotoxin adsorption and histone antagonism properties, rendering it both effective and safe for use in septic rabbits.