Aims：To establish a novel model of mechanical loading-induced knee osteoarthritis (KOA) and explore its regulatory mechanisms through transcriptomics. Methods：Knee joints of Sprague-Dawley (SD) rats were mechanically loaded with 13 N, 20 N and 27 N for 2 or 4 weeks to construct mechanically-induced KOA model. Immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, Safranin O/fast green staining, enzyme-linked immunosorbent assay (ELISA), micro-computed tomography (Micro-CT) and behavioral analysis were used to evaluate damage on the right knee joint. Transcriptomic analysis combined with validation experiments were performed to explore the regulatory mechanism of excessive mechanical loading on KOA development. Results：A vertical load of 27 N resulted in calf fractures, whereas a 13 N load did not cause remarkable pathological alteration in the knee joint. Notably, applying compression at a load of 20 N for 4 weeks (w) significantly promoted the levels of pro-inflammatory factors IL-6, IL-β and TNF-α in serum and joint fluids and markedly minimized the levels of anti-inflammatory factors IL-10 and TGF-β. Immunohistochemistry, qRT-PCR and Western blot analyses suggested that the 20 N load reduced the expression of anabolism markers (ACAN and COL2A1) and escalated the expression of catabolism markers (MMP13 and ADAMTS4). KEGG analysis and validation results showed that the PIEZ1 channel, Ca2+ signaling pathway, PI3K-AKT signaling pathway and NF-κB signaling pathway were significantly activated in the 20N-4 w group. Conclusion：Continuous loading with 20 N for 4 weeks can induce significant OA-like damage to the cartilage of the right knee in rats, which might be induced through the piezo1-Ca2+/PI3K-AKT/ NF-κB signaling pathway. Overall design: Seventy 6-week-old male SD rats were randomly classified into 7 groups: Control group ; 13 N - 2 w group; 13 N - 4 w group; 20 N - 2 w group; 20 N - 4 w group; 27 N - 2 w group; 27 N - 4 w group. A ZwickRoell static material testing machine was used for axial compression of the right knee and ankle joints of anaesthetized rats. The compression was done every two days for 2 or 4 weeks. Each compression consisted of 40 cycles and each cycle lasted for 10 seconds (s), with a base load of 5 N for 9 s and peak loads of 13 N, 20 N, or 27 N for 0.5 s. Transcriptome sequencin was performed from the right knee cartilage of rats in the blank group (n=3) and 20N - 4 w group (n=3).