The aim of this study was to determine the optimal sterilization procedure for biodegradable polyester-based guided bone regeneration membranes. The effects of sterilization using low-temperature hydrogen peroxide gas plasma, 75% ethanol (EtOH; two soaking times), and ultraviolet radiation on the structure and biological properties of electrospun poly(epsilon-caprolactone) membranes were investigated. The results demonstrated that all were effective sterilization methods. The membranes were then assessed for surface structure, wettability, and in vitro cellular responses including osteogenic differentiation by seeding with pre-osteoblasts (MC3T3-E1 cells). The cells grew well on all the sterilized membranes. The low-temperature hydrogen peroxide gas plasma-sterilized membranes, which exhibited significantly improved hydrophilicity (p<0.05), were better for cell osteogenic differentiation compared to the membranes sterilized by other methods. In addition, the cell behavior on the membranes sterilized by EtOH was superior to those sterilized by ultraviolet radiation. Finally, EtOH soaking time appeared to influence cell behavior. The results suggested that low-temperature hydrogen peroxide gas plasma treatment is the most promising method to sterilize electrospun poly(epsilon-caprolactone) membranes for guided bone regeneration.