Simple SummaryGlycosylation is an important post-translational modification made on mammalian proteins and lipids. In cancer cells, the disruption of several glycosylation patterns, such as the O-glycosylation, has been observed. The expression of incomplete/truncated O-glycans in cancer cells occurs in both solid and liquid tumors and is correlated with poor prognosis and tumor progression. The employment of monoclonal antibodies (mAbs) targeting truncated O-glycans in cancer cells could serve as an effective strategy to counteract tumor growth. In previous studies, we reported that the IgG1-humanized mAb NEO-201 binds specifically to tumor-associated variants of CEACAM5 and CEACAM6 expressed by colon, ovarian, pancreatic, non-small cell lung, head and neck, cervical, uterine and breast cancers but is not reactive against most normal tissues. Since CEACAMs are highly glycosylated proteins, in this article, we evaluated whether the epitope recognized by NEO-201 is an O-glycan. This study demonstrated that NEO-201 binds to core 1 O-glycans and targets and kills cancer cells expressing core 1 and extended core 1 O-glycans. Usually, GalNAc residue can be added on to threonine and serine to form O-glycans, suggesting that NEO-201 binds to core 1 and extended core 1 O-glycans attached to any protein carrying amino acid regions containing serine and threonineTruncated O-glycans expressed in cancer cells support tumor progression, and they may serve as potential targets to improve the monitoring and treatment of cancers. Previously, we reported that NEO-201 binds to several tumors expressing tumor-associated CEACAM5 and CEACAM6 variants but does not bind to those expressed in healthy tissues. This specific binding may be associated with the presence of truncated O-glycans attached on the protein sequence of these variants. To evaluate the glycosylation pattern targeted by NEO-201 we performed an O-glycan array consisting of 94 O-glycans. O-glycan profiles were elucidated from the human pancreatic cancer cell line CFPAC-1, human hematological neoplastic cells (HL-60, U937, K562) and human neutrophils. The O-glycan array analysis showed that NEO-201 interacts with core 1-4 O-glycans and that the binding to a specific core 1 O-glycan was the strongest. The O-glycan profiling of the NEO-201-reactive cells CFPAC-1, HL-60, U937 and human neutrophils showed that cells recognized by NEO-201 express mostly core 1 and/or extended core 1 O-glycans. In addition, NEO-201 mediates antibody-dependent cell-mediated cytotoxicity (ADCC) against tumor cells expressing core 1 or extended core 1 O-glycan profiles. These results demonstrated that NEO-201 binds to core 1 and extended core 1 O-glycans expressed in its target cells. Since GalNAc residue can be added onto threonine and serine to form O-glycans, it is very likely that NEO-201 recognizes these O-glycans attached to any protein with amino acid regions containing serine and threonine.