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<b>AIM:</b>To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2) on untransfected human corneal endothelial cells (HCECs) <i>in vitro</i>.<b>METHODS:</b> After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.<b>RESULTS:</b>TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L) altered HCE cell morphology after treatment for 36h, increased the mean optical density (<i>P</i>&lt;0.01) and the length of F-actin, reduced the mean optical density (<i>P</i>&lt;0.01) of the collagen type IV in extracellular matrix (ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72h. <b>CONCLUTION:</b>TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.