Myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), also known as C-type lectin domain family 5, member A, is a type II transmembrane protein belonging to the C-type lectin family and associates with DAP12 (also called KARAP or TYROBP). It has been reported that two isoforms of MDL-1—long form (MDL-1L) and short form (MDL-1S)—exist in mice. Previously, we observed the marked induction of MDL-1 mRNA expression during the pulmonary mycobacterial infection in mice. The data suggested that the MDL-1-expressing cells were involved in immune responses against mycobacterial infection; however, little is known about the function of MDL-1 as yet. In this study, we demonstrated the significant protein expression of MDL-1L and MDL-1S in mouse neutrophils and macrophages. MDL-1L was highly glycosylated by N-linked glycan and sialic acid. Interestingly, the expression pattern of MDL-1 was different between neutrophils and macrophages. MDL-1 expression was notably induced during the differentiation of the mouse myeloid cell line 32Dcl3 into neutrophils. Additionally, we observed that MDL-1 stimulation induced a significant amount of RANTES and macrophage-derived chemokine production in 32Dcl3 cells in cooperation with signaling through TLR. MDL-1 stimulation also up-regulated CD11b expression and maintained cell survival. Our findings indicate that MDL-1, therefore, plays an important role in immune defense as a result of an innate immunity, which involves neutrophils and macrophages.