Sigmodontinae is a very rich clade of rodents that is widespread throughout the Neotropics. The arboreal mice Juliomys comprise a poorly known branch, with incomplete information about species richness, phylogenetic position, and geographic distribution. Based on a sample from the Atlantic Forest of Southern Brazil, we name and describe a new species for the genus. This new species can be distinguished from others in the genus by its karyotype, morphological traits, and cytochrome b (Cytb) gene sequence. It has a unique karyotype (2n = 32, FN = 48), and forms a well-supported monophyletic haplogroup, which is phylogenetically distant from the remaining species of Juliomys. The genetic differentiation ranges from 11.1% to 19.7%, and there are 24 molecular autopomorphies in the Cytb gene. The new species can be distinguished from J. pictipes by morphological and morphometric analyses. However, we consider the new species as cryptic, not easily recognized by morphological characteristics of other species of Juliomys. This taxon seems to be endemic to the Brazilian Araucaria Forest in the Atlantic Forest biome, occurring in sympatry with its congeneric species J. ossitenuis and J. pictipes. In this research, we describe a new species for the genus and extend species distributions in the Atlantic Forest.