AIM: To investigate the toxicity of the E-prostanoid 2 (EP2) receptor agonist, butaprost against human subconjunctival (Tenon’s capsule) fibroblasts, and to determine the underlying mechanism. METHODS: We isolated Tenon’s fibroblasts from the subconjunctival area of healthy subjects and evaluated the types of EP receptors expressed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The toxicity of butaprost against the fibroblasts was evaluated using methyl thiazolyl tetrazolium and lactic dehydrogenase assays. The inhibition of conjunctival fibroblast proliferation by butaprost was assessed by measuring α-actin levels. The underlying mechanism was assessed by measuring intracellular cyclic adenosine monophosphate (cAMP) levels. Intergroup differences were statistically analyzed using an independent t-test. Densitometry of the Western blot bands was performed using the Image J software. RESULTS: Quantitative real-time RT-PCR revealed that the fibroblast EP2 receptor levels were higher than those of the other EP receptors. Butaprost did not show toxicity against Tenon’s tissue, but inhibited conjunctival fibroblast proliferation by reducing collagen synthesis. EP2 receptor activation enhanced the cAMP cascade, which might be an important mechanism underlying this effect. CONCLUSION: Butaprost effectively reduces the subconjunctival scarring response. Given the significance of wound healing modulation in blebs, butaprost’s inhibitory effect on subconjunctival Tenon’s fibroblasts may be beneficial in managing postoperative scarring in glaucoma surgery.