CD300LG is a novel O-glycosylated member of the CD300 antigen-like family. Besides a classical mucin-like domain, it contains a V-type Ig domain. CD300LG binds lymphocyte L-selectin via its Ig domain and supports lymphocyte rolling via its mucin-like domain. The unique structure and function of CD300LG suggest it may play an important role in inflammation. For preparation of a monoclonal antibody (MAb) against human CD300LG, prokaryotic and eukaryotic expressing human CD300LG proteins were used as immunogen and detection antigen, respectively. One stable strain of hybridomas (3C7C5A6) was successfully established using the hybridoma technique. The Western blot and immunohistochemistry analyses demonstrated that the MAb was directed against human CD300LG with high specificity. This antibody could possibly facilitate studies on the pathomechanism of inflammation and may have the potential to be a means of effective anti-inflammation.