We describe a new species of the cricetid rodent Oxymycterus (Sigmodontinae), which inhabits a transitional area between the southern Cerrado and the Atlantic Forest in south-southeastern Brazil. Compared to other Oxymycterus, the new species is large-sized with a tawny-brown pelage coloration. The new species could be differentiated from other Oxymycterus species by a combination of cranial characteristics that includes markedly large and inflated auditory bulla; a narrow rostrum and large incisive foramen, with the posterior extremity reaching the posterior region of the M1 protocone or hypoflexus; a wide parapterygoid fossa; the presence of a foramen ovale in the posterior region of the parapterygoid plate; and a thin hamular process of the squamosal. Bayesian analysis based on the mitochondrial and nuclear genes (cytochrome-b and acid phosphatase type V—Intron 2, respectively) recovered from the Oxymycterus sp. nov. showed it to be phylogenetically closely related to O. amazonicus and O. delator, all three species associated with open vegetation. The lineage leading to this clade likely emerged around 1.14 million years ago during the Early-Middle Pleistocene. Genetic distances between the new taxa and these two species calculated from comparison of cytochrome-b sequences are 3.7% and 4.1%, respectively. Currently, Oxymycterus sp. nov. is known from only two unprotected sites, with the type locality inserted in an area under the process of conurbation. Our study raises the number of living species in the genus to 16.