Twelve flavonoid glycosides including five undescribed ones, lepisativutimines Q-U, were isolated and identified from Lepidium sativum seeds. Acid hydrolysis followed by acetic derivatization and GC analysis were conducted to determine the absolute configurations for sugars. Lepisativutimine U and beitingxinhuangtong A were uncommon kaempferol 8-S-glycosides, and complete NMR data of beitingxinhuangtong A were provided for the first time. Mass fragmentation pathways of isolated compounds were deduced based on their MS/MS data, and characteristic mass fragmentation patterns were summarized. Lepisativutimine U and kaempferol-7-O-α-l-rhamnopyranoside exhibited weak anti-diabetic activity against PTP1B with IC