We have previously reported on a functionalized folic acid (FA) conjugated poly(styrene-alt-maleic anhydride) (SMA) via biological linker 2,4-diaminobutyric acid (DABA) (FA-DABA-SMA) copolymer. This biocompatible nanocopolymer self-assembles in a pH-dependent manner, providing stimuli responsiveness, active targeting, and extended release of hydrophobic chemotherapeutic agents and effectively penetrates the inner core of 3-dimensional cancer spheroid models. The empty FA-DABA-SMA decreased tumor spheroid volume, revealing a previously unknown mechanism of action. Here, we investigated the potential mechanism of the small (20 kDa) and large (350 kDa) FA-DABA-SMA empty copolymers affecting the folic acid receptor alpha (FRα) signaling properties in breast and prostate cancer cell lines. Microscopic imaging, immunocytochemistry, flow cytometry, Caspase 3/7 apoptosis assays, Incucyte live cell tracking, the scratch wound assay, the water-soluble tetrazolium salt-1 (WST-1) cell viability assay, morphologic changes, and Western blot for the expression levels of FRα on the cell surface were used on MDA MB-231 and MCF-7 breast and DU-145 prostate cancer cell lines. The findings indicate that FA-DABA-SMA increases FRα expression levels in breast MDA MB-231 cancer cells and then disrupts FR signaling by reducing HES1 and NOTCH1 protein expression levels. Also, FA-DABA-SMA induces apoptosis and further causes a change in the MDA MB-231 cells' morphology and significantly reduces their ability to migrate in a scratch wound assay. Collectively, these findings provide a novel insight into the functionalized FA-DABA-SMA copolymer. The 350 and 20 kDa copolymers actively target FRα to initialize internationalization. However, only the large size and sheet-shaped 350 kDa copolymers disrupt FRα signaling. The significance of these novel findings reveals that the copolymer's intracellular activity is critically dependent on the size and structural shape. This report offers novel therapeutic insight into a dual mechanism of the FA-DABA-SMA copolymer for its therapeutic potential to treat cancer.