BACKGROUND Understanding the biological features and developmental progress of cervical cancer is crucial for disease prevention. This study aimed to determine the nanomechanical signatures of cervical samples, ranging from cervicitis to cervical carcinomas, and to investigate the underlying mechanisms. MATERIAL AND METHODS Forty-five cervical biopsies at various pathological stages were subjected to atomic force microscopy (AFM) measurements. Cdc42 and collagen I were quantified using immunohistochemical staining to investigate their relationship with nanomechanical properties of cervical cancers and premalignant lesions. RESULTS We found that the lower elasticity peaks (LEPs) in the high-grade squamous intraepithelial lesion (HSIL) group (21.24±3.83 kPa) and higher elasticity peaks (HEPs) in the cancer group (81.23±8.82 kPa) were upshifted compared with the control group (LEP at 8.51±0.18 kPa and HEP at 44.07±3.54 kPa). Furthermore, compared with the control [29.51±13.61 for cell division cycle 42 (Cdc42) expression and 28.61±17.65 for collagen I expression], immunohistochemical staining verified a significant increase of Cdc42 in the HSIL group (50.57±23.85) and collagen I (56.09±25.70) in the cancer group. In addition, using the Pearson correlation coefficient, Cdc42 expression tended to be positively correlated with LEP locations (r=0.63, P=0.012), while collagen I expression displayed a strong and positive correlation with HEP positions (r=0.88, P<0.001). CONCLUSIONS The nanomechanical properties of HSIL and cancer biopsies show unique features compared with controls, and these alterations are probably due to changes in cytoskeleton and extracellular matrix contents.