Four ring-A seco-cucurbitane triterpenoids, colocynthenins A-D (1-4), together with seven known cucurbitane triterpenoids (5-11), were isolated from the fruits of Citrullus colocynthis. Their structures and absolute configurations were elucidated based on spectroscopic analysis and quantum chemical ECD calculations. Compound 1 possesses an unprecedented 2,11-lactone moiety, while compound 2 is the first reported cucurbitane triterpenoid with an unusual cyano group. Compounds 1 and 3 showed acetylcholinesterase inhibitory activities in a standard in vitro assay, with IC50 values of 2.6 and 3.1 μM, respectively.