Activated protein C (APC) can exert two major distinct activities: 1) anticoagulant activity, via inactivation of factors Va and VIIIa, and 2) cytoprotective activity including anti-apoptotic and anti-inflammatory activities mediated by two receptors, Endothelial Protein C Receptor (EPCR) and Protease Activated Receptor-1 (PAR-1). Interest in APC’s receptor-mediated effects is sparked by APC’s reduction of mortality in severe sepsis patients and by the beneficial neuroprotective activities of APC on brain endothelium, on neurons and in rodent ischemic stroke models. Our previous targeted mutagenesis of exosite residues in the protease domain in APC’s heavy chain proved that anticoagulant activity can be almost ablated while leaving anti-apoptotic activity intact, thus showing that some heavy chain exosite residues are required for one activity but not the other. To further characterize APC exosites required for various functional activities, we extended site-directed mutagenesis studies to the C-terminal region of the light chain (residues 142–155) which was indirectly implicated as an anticoagulant exosite but which has not been targeted using mutagenesis. Alanine-scanning mutagenesis was used to probe the requirement for Arg143, Lys146, Arg147, Glu149, Lys150, Lys151 and Arg152 for zymogen activation and for expression of APC’s anticoagulant and cytoprotective activity. Kinetic data for activation of each mutated zymogen by thrombin and by thrombin-thrombomodulin were indistinguishable from wild type protein C results. APC mutants with single Ala substitutions at residues 146, 147, 150 and 151 had significantly reduced anticoagulant activity in APTT assays while substitution at 143 and 152 showed little change relative to wild type APC. Surprisingly, the Ala149-APC exhibited over 3-fold increased anticoagulant activity. No mutation altered the half-life of APC amidolytic activity in plasma, suggesting that APC’s reaction with key serine protease inhibitors was unaltered by these mutations. As determined using staurosporine-induced endothelial cell apoptosis assays, replacement of the various Arg and Lys residues by Ala had little significant effect on APC cytoprotective activity; however, the Glu149 to Ala substitution markedly reduced APC cytoprotective activity. Thus, whereas this latter mutation increased by 3-fold anticoagulant activity, it diminished APC direct effects on cells. In summary, these studies of the C-terminus of the APC light chain emphasize the distinction between exosite residue requirements for APC’s anticoagulant versus cytoprotective mechanisms; furthermore, these results may contribute to the design of safer therapeutic APC variants with reduced risk of bleeding due to reduction in anticoagulant activity but with the normal beneficial direct effects of APC on cells.