Information about the mechanical properties of cancer cells leads to new insights about their malignancy levels. The more flexible the cancer cells and their nuclei are, the more aggressive and invasive they are. Flexibility is a result of composition and properties of molecular constituents of cells and its extent is expressed by deformation. Differences in the mechanical properties could be modulated by topography and chemistry of the substrate. In this study, the main hypothesis is that the difference in the mechanical properties of malignant and benign breast cancer cells could be used as a discriminator of these cells and reflected by the extent of nuclear deformation on micropatterned substrates. We compared benign (MCF10A), malignantnoninvasive (MCF7), and malignant highly invasive (MDAMB231) breast cancer cell lines using their nuclear deformability on micropatterned surfaces designed with square prism-shaped micropillars of poly(methyl methacrylate) (PMMA) (8 μm high, 4 × 4 μm2 area, 4 μm gap). Several shape descriptors (circularity, solidity, roundness, aspect ratio) were used to analyze nuclear deformation. We were able to discriminate the three cells when the descriptor circularity and hydrophobic micropatterned surfaces were used. The cells showed nuclear deformability in the order following the extent of their malignancies. The most aggressive cell, MDAMB231, had the lowest circularity value, 0.37, whereas the noninvasive malignant, MCF7, and benign, MCF10A, cells had higher values 0.47 and 0.77, respectively. Mechanism of the deformation was shown at the molecular level that the expression of Lamin A/C and Nesprin-2 genes decreased with increased nuclear deformation. In summary, biomechanical properties of cells can provide useful information about their cancer state and they can be reflected in the biological markers.