Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology.