Speciation is a fundamental evolutionary process, which results in genetic differentiation of populations and manifests as discrete morphological, physiological as well as behavioral differences. Each species has had its own evolutionary trajectory, formed by many types of selection pressures and random drift, making it extremely complicated to associate genetic differences between the species with the phenotypic differences. In the present study, we have used an in vitro model to analyze in depth the genetic and gene regulation differences between fibroblasts of two closely related mammals, arctic/subarctic mountain hare (Lepus timidus Linnaeus) and a temperate, steppe-like climate adapted brown hare (Lepus europaeus Pallas). We discovered the existence of a species-specific expression pattern of 1,623 genes, manifesting in differences in cell growth, respiration, and metabolism. Interspecific differences in the housekeeping functions of fibroblast cells suggest speciation acts on fundamental processes, even in these two interfertile species. Our results help to understand the molecular constituents of a species difference on a cellular level, which could contribute to the maintenance of the species boundary.