Podoplanin (PDPN/Aggrus/T1α), a platelet aggregation-inducing type I transmembrane sialoglycoprotein, is involved in tumor invasion and metastasis. Furthermore, podoplanin expression was reported to be involved in poor prognosis of several cancers. Although many anti-podoplanin monoclonal antibodies (MAbs), such as NZ-1 and D2-40, have been established, those epitopes are limited to platelet aggregation-stimulating (PLAG) domain of podoplanin. In this study, we developed and characterized a novel anti-podoplanin MAb, LpMab-7, that is more sensitive than NZ-1 in immunohistochemistry. We identified the minimum epitope of LpMab-7 as Arg79-Leu83 of human podoplanin, which is different from PLAG domain, using ELISA, Western blot analysis, and flow cytometry. Because LpMab-7 has high sensitivity against podoplanin, LpMab-7 is expected to be useful for molecular targeting therapy for podoplanin-expressing cancers.