The mucin‐type sialoglycoprotein podoplanin (aggrus) is involved in tumor cell‐induced platelet aggregation and tumor metastasis. C‐type lectin‐like receptor‐2 (CLEC‐2) was recently identified as an endogenous receptor of podoplanin on platelets. However, the pathophysiological importance and function of CLEC‐2 have not been elucidated. Here we clarified the pathophysiological interaction between podoplanin and CLEC‐2 in vitro and in vivo. Using several deletion mutants of CLEC‐2 expressed as Fc chimeras, we first identified an important podoplanin‐recognition domain in CLEC‐2. Furthermore, the podoplanin–CLEC‐2 interaction was confirmed using several deletion mutants of podoplanin expressed as Fc chimeras. Not only the disialyl‐core1‐attached glycopeptide but also the stereostructure of the podoplanin protein was found to be critical for the CLEC‐2‐binding activity of podoplanin. We next synthesized various glycopeptides of podoplanin that included both the platelet aggregation‐stimulating domain and O‐glycan on Thr52. Interestingly, a disialyl‐core1‐attached glycopeptide was recognized specifically by CLEC‐2. Moreover, the anti‐podoplanin monoclonal antibody NZ‐1 suppressed both the podoplanin–CLEC‐2 interaction and podoplanin‐induced pulmonary metastasis, suggesting that CLEC‐2 is the first pathophysiological receptor of podoplanin to be identified. In summary, we clarified the molecular interaction in vitro and in vivo between a platelet aggregation‐inducing factor, podoplanin, and its specific pathophysiological receptor on platelets, CLEC‐2. Podoplanin and CLEC‐2 might represent promising therapeutic targets in cancer metastasis. (Cancer Sci 2008; 99: 54–61)