Protease-activated receptor 2 (PAR2) is a GPCR activated by proteolytic cleavage of its extracellular N-terminus to reveal a tethered ligand (SLIGKV) that folds back and self activates the receptor. Peptide agonists are widely used to investigate PAR2 activation and signalling pathways. While there are various PAR2 agonists based on the canonical human tethered ligand sequence, one of the most common is 2-furoyl-LIGRLO-NH2. This selective PAR2-activating peptide shows high potency, selectivity and stability in cultured cells and in vivo models. The aim of this study was to determine the role of PAR2 activation in colon cancer cells. Among the 33 different human cancers, colon cancer shows the highest PAR2 expression and was significantly upregulated in human colon cancer tissues versus normal colon tissues. A common chemotherapeutic drug, doxorubicin, used to treat different cancers in the clinic is facing wide-spread drug resistance. The majority of colon cancer patients now develop resistance to doxorubicin treatment. New and effective targeted therapies are required to overcome chemoresistance and decrease the failure rate of treatment. We found that 2f-LIGRLO-NH2 induced PAR2 activation and attenuated doxorubicin-induced cell death, production of reactive oxygen species (ROS) and caspase activity in human colon cancer HT29 cells. We were interested in how peptide-activation of PAR2 affects drug treatment. We showed that 2f-LIGRLO-NH2 activated PAR2 that signals through ERK1/2 phosphorylation, which was linked here to upregulation of anti-apoptotic MCL-1 and Bcl-xL proteins. Either a PAR2 antagonist or an ERK1/2 inhibitor could restore the effects of doxorubicin in inducing cell death, ROS production and caspase levels in HT29 colon cancer cells. Collectively, this study suggests that peptide activated PAR2 may result in pro- or antiapoptotic signalling mechanisms that control resistance to doxorubicin in colon cancer cells and thus may represent a possible drug target in colon cancer.