Stratum corneum intercellular lipids play an important role in skin barrier function and are therefore considered useful in pharmaceutical and cosmetics products. In this study, we formulated cholesterol (Cho), the main component of intercellular lipids, into bicelles using a semi-spontaneous method, with the ultimate aim of extending the utility of bicelles in cosmetics. Bicelles are disk-shaped structures in which the planar part comprises long-chain phospholipids and the rims comprise short-chain phospholipids. We used soybean lecithin (SL) and nonionic surfactants (NS) to formulate Cho into bicelles using a semi-spontaneous method. Cho was successfully formulated into bicelles using poly(oxyethylene) cholesteryl ether (with an average of 10 ethylene oxide units, ChEO10) as the NS. We identified the boundaries of the bicelle region in the SL-Cho-ChEO10 system by measuring particle sizes. A bicelle region was formed up to around a maximum mixing ratio of 10wt% Cho, representing a mole fraction (mol%) of approximately 33. Phase changes in the system were observed. Comparison of poly(oxyethylene) cholesteryl ethers containing an average of 10 or 20 (ChEO20) ethylene oxide units revealed that the size of the hydrophilic group affects both the bicelle formation region and the mixing ratio of Cho. We analyzed the state of the SL-Cho-ChEO10 bicelles by differential scanning calorimetry and found that samples containing only phospholipid (SL/Ch/ChEO10 = 1/0/0) provided an endothermic peak between 50 and 55 °C, corresponding to the gel-liquid crystal transition point of the phospholipid. In contrast, the bicelle regions (SL/Cho/ChEO10 = 0.4/0/0.6 and 0.37/0.05/0.58) lacked a gel liquid crystal transition point and a liquid-ordered state was induced by Cho or ChEO10. This indicates that these bicelles are highly stable towards thermal changes.