Increased tissue rigidity is an emerging hallmark of cancer as it plays a critical role in promoting cancer growth. However, the field lacks a defined characterization of tissue rigidity in dual-stage carcinogenesis of lung squamous cell carcinoma (SCC) in vivo. Pre-malignant and malignant lung SCC was developed in BALB/c mice using N-nitroso-tris-chloroethylurea (NTCU). Picro sirius red staining and atomic force microscopy were performed to measure collagen content and collagen (diameter and rigidity), respectively. Then, the expression of tenascin C (TNC) protein was determined using immunohistochemistry staining. Briefly, all tissue rigidity parameters were found to be increased in the Cancer group as compared with the Vehicle group. Importantly, collagen content (33.63 ± 2.39%) and TNC expression (7.97 ± 2.04%) were found to be significantly higher (< 0.05) in the Malignant Cancer group, as compared with the collagen content (18.08 ± 1.75%) and TNC expression (0.45 ± 0.53%) in the Pre-malignant Cancer group, indicating increased tissue rigidity during carcinogenesis of lung SCC. Overall, tissue rigidity of lung SCC was suggested to be increased during carcinogenesis as indicated by the overexpression of collagen and TNC protein, which may warrant further research as novel therapeutic targets to treat lung SCC effectively.