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Modified citrus pectin (MCP), a commercially available dietary supplement prepared from citrus pectin, contains several different polysaccharide domains, but its primary chemical structure and the binding epitopes that antagonize galectin-3 function remain unclear. In this study, five fractions were isolated from MCP after endo-polygalacturonase degradation (EMCP) and a combination of DEAE-cellulose and Sepharose CL-6B or Sephadex G-75 chromatography. Their primary structures, abilities to inhibit galectin-3-mediated hemagglutination, and antiproliferation activities on MCF-7 and A549 cell lines were studied. Results showed that EMCP-3p, one of the five fractions, was composed of Glc (89.8%), Gal (3.8%), Ara (3.1%), GalA (1.1%), Man (0.9%), and Rha (1.3%) with an average molecular weight of 88.4 KDa, which had the most substantial degree of galectin-3 inhibition with an MIC of 31.25 μg/mL, and it exhibited remarkable cytotoxicity against MCF-7 (36.7%) and A549 (57.4%) cell lines. These results provide new insight into the structure–function relationships of EMCP-derived polysaccharides.