Podoplanin (PDPN) induces cell invasion and cancer metastasis, and its expression in cancer cells or cancer-associated fibroblasts has been reported to be involved in poor prognosis of several cancers including malignant gliomas and lung cancers. PDPN is also expressed in normal cells such as lymphatic endothelial cells, lung type I alveolar cells, and kidney podocytes. Many anti-PDPN monoclonal antibodies (MAbs) have been established; however, almost all anti-PDPN MAbs recognize a platelet aggregation-inducing (PLAG) domain, because the PLAG domain is known to be highly immunogenic. Here, we developed and characterized LpMab-9, a novel anti-PDPN MAb. LpMab-9 reacted with LN319 glioblastoma cells, but did not react with LN319/PDPN knock-out cells. LpMab-9 showed slight reaction with sialylated O-glycan-deficient PDPN. We identified the minimum epitope of LpMab-9 as Thr25-Asp31, which is the N-terminus of human PDPN, using Western blot analysis. Furthermore, Thr25, Gly26, Gln27, and Pro28 were shown to be critical for LpMab-9-binding to PDPN using flow cytometry. Antibody-overlay lectin microarray using LpMab-9 demonstrated that PDPN reacts with sialic acid ± core1 binders and sialo-mucin binders. Taken together, these results indicate that LpMab-9 recognizes O-glycosylation of Thr25 in the N-terminus of PDPN. LpMab-9 could be useful for uncovering the physiological function of O-glycosylated N-terminus of human PDPN.