The seeds of Ziziphus mauritiana Lam. have been traditionally used for treatment of various complications including insomnia and anxiety. They are popularly used as sedative and hypnotic drugs in China, Korea, Myanmar, Vietnam, and other Asian countries. However, no scientific proof on hypnotic activity of Z. mauritiana seeds (ZMS) was reported. In this study, the hypnotic activity of 50% ethanolic extract from ZMS was observed on the loss of righting reflex in mice using pentobarbital-induced sleep mice method. The contents of total phenolics and total flavonoids in the extract were also determined. The results showed that the 50% ethanolic extract from ZMS contained total phenolics 27.62 ± 1.43 mg gallic acid equivalent (GAE)/g extract and total flavonoids 0.74 ± 0.03 mg quercetin equivalent (QE)/g extract. Oral administration of the extract at the dose of 200 mg/kg significantly increased the sleeping time in mice intraperitoneally administered with sodium pentobarbital (50 mg/kg body weight). These results supported the traditional use of ZMS for the treatment of insomnia. The seeds of Z. mauritiana should be further developed as an alternative sedative and/or hypnotic product.