Background and Objective To investigate the cytotoxic effects of silicone oil on the cultivated human corneal endothelial cells (CEs). Methods We cultured human CE and passed them in insert wells that allowed the apical side of CE monolayer in contact with the silicone oil. The tested silicone oils were of two different viscosities, 1000 and 5000 centistoke (CS). MTS proliferation bioassay and calcein-acetoxymethyl ester (CAM)-ethidium homodimer staining were performed to evaluate cell viability after CEs were co-cultured with silicone oils for 48 h. Apoptosis of CEs was evaluated by TdT-mediated dUTP nick-end labelling (TUNEL) stain. Results The MTS bioassay showed that contact of silicone oil inhibited CE proliferation. The higher viscosity (5000 CS) silicone oil suppressed cell cycling significantly more than the lower viscosity (1000 CS) silicone oil. CAM-ethidium homodimer staining revealed CE death, 9.1 +/- 0.1% (1000 CS silicone oil) and 41.6 +/- 0.4% (5000 CS), but apoptosis played only minor role in silicone oil toxicity, 1.7 +/- 0.1% (1000 CS silicone oil) and 9.4 +/- 0.1% (5000 CS). Conclusions Silicone oil is cytotoxic to cultivated human CEs. Avoiding the forward migration of silicone oil to the anterior chamber and corneal CE contact is critical in preventing silicone oil-associated keratopathy. Silicone oil should be removed as early as possible once the goal of tamponade has been achieved.