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Background and Aims. Hepatocellular carcinoma is an aggressive malignancy of the liver and is ranked as the sixth most common cancer worldwide. There is still room for novel markers to improve the diagnosis and monitoring of HCC. Our observations in cancer databases that PLXNC1 is upregulated in HCC led us to investigate the expression profile of Plexin C1 mRNA and protein in HCC cell lines and tissues. Methods. A recombinant protein encompassing part of the extracellular domain of Plexin C1 was used as an antigen for monoclonal antibody development. Transcript and protein levels of Plexin C1 in HCC cell lines were determined by RT-qPCR and Western blotting, respectively. In vivo evaluation of Plexin C1 expression in HCC tissues was accomplished by immunohistochemistry studies in tissue microarrays. Results. A monoclonal antibody, clone PE4, specific to Plexin C1, was generated. In silico and in vitro analyses revealed a Plexin C1-based clustering of well-differentiated HCC cell lines. Staining of HCC and nontumoral liver tissues with PE4 showed a membrane-localized overexpression of Plexin C1 in tumors (p=0.0118). In addition, this expression was correlated with the histological grades of HCC cases. Conclusions. Plexin C1 distinguishes HCC cells of epithelial characteristics from those with the mesenchymal phenotype. Compared to the nontumoral liver, HCC tissues significantly overexpress Plexin C1. The newly generated PE4 antibody can be evaluated in larger HCC cohorts and might be exploited for the examination of Plexin C1 expression pattern in other epithelial malignancies.