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Simple Summary Cold shock Y-box binding protein-1 plays a crucial role in cancerous cell transformation and proliferation. Experimental evidence links autoantibody formation with cancer diseases as well as YB 1 protein levels. Hence, we investigated autoantibody formation targeting YB-1 in cancer patients. Using recombinant proteins and specific peptide arrays, we mapped linear epitopes, which localize in the cold shock and C-terminal domain of the protein, in cancer patients that differ from healthy controls. Furthermore, cancer sera containing autoantibodies that target YB-1 extend the half-life of the YB-1 protein. Since extracellular YB-1 serves as a ligand for receptor Notch3 as well as TNFR1, this may contribute to aberrant signaling that promotes tumor development. In the clinical setting, we envision setting up detection assays for the immune response against YB-1, which may aid in screening for cancer. Abstract Cold shock Y-box binding protein-1 participates in cancer cell transformation and mediates invasive cell growth. It is unknown whether an autoimmune response against cancerous human YB-1 with posttranslational protein modifications or processing develops. We performed a systematic analysis for autoantibody formation directed against conformational and linear epitopes within the protein. Full-length and truncated recombinant proteins from prokaryotic and eukaryotic cells were generated. Characterization revealed a pattern of spontaneous protein cleavage, predominantly with the prokaryotic protein. Autoantibodies against prokaryotic, but not eukaryotic full-length and cleaved human YB-1 protein fragments were detected in both, healthy volunteers and cancer patients. A mapping of immunogenic epitopes performed with truncated E. coli-derived GST-hYB-1 proteins yielded distinct residues in the protein N- and C-terminus. A peptide array with consecutive overlapping 15mers revealed six distinct antigenic regions in cancer patients, however to a lesser extent in healthy controls. Finally, a protein cleavage assay was set up with recombinant pro- and eukaryotic-derived tagged hYB-1 proteins. A distinct cleavage pattern developed, that is retarded by sera from cancer patients. Taken together, a specific autoimmune response against hYB-1 protein develops in cancer patients with autoantibodies targeting linear epitopes.