This study was focused on the problem of regulation of the proinflammatory activity of mast cells through a specific class of protease-activated receptors, PAR1. The study demonstrated for the first time the regulation of the activity of RBL-2H3 cells, mast cell analogues, by a new PAR1 agonist, peptide NPNDKYEPF-amide. It has been shown that the PAR1 agonist peptide, like activated protein C (APC), exhibited anti-inflammatory and cytoprotective effects on RBL-2H3 cells under activation by pro-inflammatory factors. Incubation of mast cells with lipopolysaccharide (LPS) caused a transient increase in the concentration of intracellular free calcium, increased the level of histamine secretion by the cells, and reduced their proliferation activity. Pre-incubation of the cells with both the peptide and APC prevented the effect of endotoxin on the RBL-2H3 cells. Application of thrombin and calcium ionophore led to actin reorganization, which might indicate cell activation and initiation of secretion. Pretreatment of the cells with both the peptide and APC in the presence of activators led to the ordering of actin in the submembrane region of the cells, which was typical for the control group. Thus, the newly discovered anti-inflammatory and protective properties of the PAR1 agonist peptide open the possibility of searching for new approaches to the treatment of inflammatory processes based on drugs of a peptide nature through modulating the activity of the PAR family receptors.