The serpin, C1 inhibitor (C1INH) is the only known plasma inhibitor of C1s, the initiating serine protease of the classical pathway of complement. The complement system is vital for host immunity, but also plays a major role in inflammatory diseases. Thus, understanding the regulation of the interaction between C1s and C1INH will yield insights into the regulation of inflammatory processes. Like other serpin-protease partners, their interaction has been shown to be accelerated through their interaction with polyanions such as heparin and dextran sulphate. Polyphosphate (polyP) is a physiologically occurring polyanion that is stored in dense granules of a number of cell types involved in inflammation. Recently, polyP has been shown to bind C1-inhibitor, which raises the possibility that it may act as a cofactor of C1INH and thus act to regulate the classical pathway via this mechanism. Here, we showed that C1s also binds this polyanion and that this was mediated in part by a recently discovered exosite on the serine protease domain of the enzyme. Complex formation was increased in the presence of polyP, indicating that it does act as a cofactor for the C1s-C1INH interaction. PolyP was also shown to significantly increase the rate of interaction between C1s and C1INH, to an extent comparable or exceeding that of heparin. These effects were shown to be physiologically relevant by demonstrating that polyP enhanced C1INH inhibition of C1s-mediated cleavage of C4 in a size- and concentration-dependent manner and that C4d deposition on endothelial cells was significantly suppressed by polyp. This study indicates that polyP is a novel cofactor for the C1s-C1INH interaction and thus is an important regulator of complement activation.