〔Abstract〕 To establish an in vitro cell model of Cobalt dichloride (CoCl2)-induced epithelial-mesenchymal transition (EMT) in rat renal tubular epithelial cells (NRK-52E). Methods NRK-52E cells were cultured in vitro and randomly divided into a blank control group (NC group) and a CoCl2 treatment group. The CoCl2 treatment group underwent 1, 2, 3, and 4 cycles of treatment, with each cycle consisting of CoCl2 treatment for 9 h followed by recovery in CoCl2-free medium for 3 h. The optimal concentration and time of CoCl2-induced EMT were screened using the CCK-8 assay. Morphological changes in cells were observed using light microscopy and phalloidin staining. The expression levels of EMT marker proteins were detected by Western blot and immunofluorescence. Results Compared with the NC group, stimulation by 100 μmol/L CoCl2 for 48 h significantly induced apoptosis (P<0.01), meeting the requirements for subsequent experiments. Western blot results showed that the expression of hypoxia-inducible factor-1α (HIF-1α) and α-smooth muscle actin (α-SMA) significantly increased in the 3-cycle group treated with 100 μmol/L CoCl2 for 9 h followed by recovery for 3 h (P<0.001), indicating the most pronounced fibrotic response. Observations under light microscopy and rhodamine-labeled phalloidin staining revealed that the morphological changes and cytoskeletal rearrangement of NRK-52E cells were most significant in the 3-cycle group treated with 100 μmol/L CoCl2 for 9 h followed by recovery for 3 h, demonstrating the best model stability. Immunofluorescence results showed that compared with the NC group, the fluorescence intensity of the fibrous matrix protein Collagen I significantly increased in the 3-cycle group treated with 100 μmol/L CoCl2 for 9 h followed by recovery for 3 h (P<0.001). Conclusion The protocol involves treating NRK-52E cells with 100 μmol/L CoCl₂ for 9 h, following 3 h of recovery in CoCl₂-free medium. Repeating this cycle three times can establish an in vitro EMT model.