Mast cells (MCs) are immune cells that produce numerous immunological mediators involved in inflammatory and allergic responses. Increased numbers of MCs are observed in chronic inflammatory reactions in organs such as the colon. There, MCs seem to participate in deleterious immune responses and tissue damage, but the detailed mechanisms of their activation are not known, mostly because procedures to obtain MC primary cultures from the colonic mucosa are expensive and time-consuming and present low yield. Here we describe a protocol to obtain MCs from the colonic mucosa (cmMCs) of C57BL/6 mice with high yield, viability and purity. Mucosal colon cells were dispersed by enzymatic digestion, and cmMCs were isolated by Percoll continuous-gradient centrifugation. This method allowed for the purification of 1,446,667 ± 112,442 cell/g of mucosal tissue, with 87.22% viability and 95.16% purity. The mucosal-like phenotype was predominant in isolated cmMCs, characterized by weak toluidine blue staining but strong expression of MC protease-1 (Mcpt1). Activation assays showed that freshly isolated cmMCs increased intracellular calcium and showed degranulation in response to ATP or IgE-antigen-dependent FcεRI cross-linking. This highly reproducible technique is cost-effective and requires no specialized equipment. This protocol could be applied in research related to inflammatory bowel disease, colon cancer or food allergies.