Complement plays crucial roles in the immune system, but incorrect regulation causes inflammation and targeting of self-tissue, leading to diseases such as systemic lupus erythematosus, rheumatoid arthritis and age-related macular degeneration. In vivo, the initiating complexes of the classical and lectin pathways are controlled by SERPING1 (C1-inhibitor), which inactivates their C1s and MASP-2 protease components. Glycosaminoglycans (GAGs) and dextran sulfate (DXS) are able to significantly accelerate SERPING1-mediated inactivation of the key effector enzyme of the classical C1 complex, C1s, though the mechanism is poorly understood. Here it was shown that C1s can bind to DXS and heparin and that these polyanions could enhance C1s proteolytic activity at low concentrations and inhibit it at higher concentrations. The recent determination of the crystal structure of SERPING1 has given rise to the hypothesis that both serpin-polyanion and protease-polyanion interactions might be required to accelerate the association rate. To determine how much of this acceleration is due to protease-polyanion interactions, a chimeric mutant of alpha1-antitrypsin containing the P4-P1 residues of the SERPING1 reactive centre loop was produced. Like SERPING1, this molecule is able to effectively inhibit C1s, but is unable to bind polyanions. DXS exerted a biphasic effect on the association rate of C1s which correlated strongly with the effect of DXS on C1s proteolytic activity. Thus, while polyanions are able to bind C1s and modulate its activity, polyanion interactions with SERPING1 must also play a vital role in the mechanism by which these cofactors accelerate the C1s/SERPING1 reaction.