NK cell markers and receptors have been discovered in many mammalian species, such as humans, mice, rats, pigs, and cows. However, there is still a lack of information concerning NK cell markers or receptors in canines. We have discovered that canine CD5-low density (CD5lo) cells in PBL are closely associated with NK cell characteristics. CD5lo cells comprised 14.9 ± 6.68% of the total PBL. A high proportion of the CD5lo cell population expressed CD3 (96.6%), CD8α (77.7%), CD8β (53%), α/β TCR (83%), and CD11/18 (80%), but the expression of γ/δ TCR (6.5%), CD4 (10.6%), and CD21 (2.4%) was low. CD5 lo cells were larger than CD5-high density (CD5hi) cells. Light and electron microscopy revealed numerous large cytoplasmic granules in CD5lo cells, especially after IL-2 stimulation, which was in contrast to CD5hi, in which intracytoplasmic granules were not frequently seen. After IL-2 stimulation, CD5lo cells had significantly stronger NK cytotoxicity than CD5hi cells. CD5lo cells had much higher mRNA levels for NKG2D, CD16, CD94, CD160, perforin, and granzyme than CD5hi. Following IL-2 stimulation, CD5lo cells had significantly higher mRNA levels of NKp30, NKp44, CD16, and CD94 than CD5 hi cells. In addition, IL-2-stimulated, CD5lo-depleted PBL showed a loss of NK cytotoxicity. CD5lo cells also showed significantly lower antigen-specific cytotoxic T cell activity as compared with CD5hi cells. Taken together, the CD5lo subset in canine PBL is closely related to canine NK cells, and CD5lo can be used as a phenotypic marker for an IL-2-dependent canine NK cell enrichment.