The dermal papilla (DP) is an indispensable mesenchymal component of the hair follicle, which regulates its morphogenesis and cycling. Although rodent DP shares fundamental biological properties with that of humans, they are distinct in some aspects, for example, response to hormones. Dogs suffer from hair loss due to hypothyroidism or androgenic alopecia, suggesting that they share some intrinsic properties with human DP cells. The aim of this study is to assess the characteristics of canine DP cells and find an appropriate condition for in vitro expansion. Similar to human DP, canine DP contains blood vessel structures, which is absent mouse DP. Isolation of intact canine DP is possible by microdissection technique, which enabled the isolation of DP total RNA and gene expression analysis. The genes known to be expressed in mouse and human DP (DP genes), such as ALP and LEF1, was overrepresented in canine DP, indicating that essential molecular mechanism that defines the intrinsic properties of DP is conserved over the species. The growth of canine DP is moderate in a conventional culture condition for mouse and human DP. Among culture mediums tested, AmnioMax (Invitrogen) enabled canine DP cells to grow at high proliferative rate. DP gene expression became moderate but still at detectable levels in AmnioMax culture. It has been reported that culture techniques such as addition of in keratinocyte-conditioned medium or use of extracellular matrix-coated dishes could ameliorate DP gene expression. However, those approaches did not significantly improved DP gene expression in cultured canine DP cells, suggesting that the effect of supplement in AmnioMax might override anticipated influences. This study opens the door to the molecular biological characterization of canine DP and provides essential information for further optimization of culture condition.