A new iridoid glycoside and three known iridoid glycosides were isolated from the aerial parts of Barleria trispinosa. The structure of the new compound was determined as 6-alpha-L-rhamnopyranosyl-8-O-acetylshanzihiside methyl ester. The known compounds were identified as 6,8-O,O-diacetylshanhiside methyl ester (acetyl barlerin), 8-O-acetylshanzhiside methyl ester (barlerin) and shanzhiside methyl ester, which were isolated from the plant for the first time. The structures of the isolated compounds were elucidated by spectroscopic evidence, mainly one- and two-dimensional proton and carbon-13 NMR spectroscopy.