Background: Ziziphi Spinosae Semen (ZSS), the seed of Ziziphus jujuba var. spinosa (Bunge) Hu ex H. F. Chow., is a traditional herb for insomnia and anxiety in eastern Asia. However, few researches have been concerned with its effect on ameliorating memory and learning performance. Objective: To investigate the constituents of ZSS water soluble extract and its ameliorating learning and memory in mice. Materials and Methods: A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed and validated to determine the main constituents in the extract. The effect of ZSS water soluble extract on memory and learning performance was investigated in mice by Y-maze and passive avoidance test. Results: The extract could significantly decrease the number of errors (NOE), and increase the transfer latency time (TLT) and electrical stimuli time (EST). In addition, spinosin, jujuboside A (JuA) and jujuboside B (JuB) were simultaneously identified and quantified in the extract. Their contents in the extract were as followed: Spinosin (223.51mg/g), JuA (63.76mg/g) and JuB (26.29mg/g). Conclusion: The extract played a promising role in ameliorating memory in mice with alcohol induced memory retrieval disorders, and might help to improve learning capacity to some extent. Spinosin, JuA and JuB were the predominant constituents, which might be mainly responsible for the definite activity.