The peptide chemical constituents and neuroprotective activity in the whole herb of Selaginella prostrata were investigated.Thin-layer in situ chemical reaction oriented separation method based on ninhydrin reagent color development was used to separate and purify the compounds by positive and reversed phase silica gel column chromatography and high performance liquid chromatography.The structures of the compounds were identified by spectral data analysis and literature comparison.Fifteen peptides compounds were isolated from 95% ethanol extract of whole herb of S. prostrata.They were identified as pulvpeptin A (1),nocardiotide A (2),dianthin C (3),longicalycinin A (4),aurentiamide (5),aurantiamide acetate (6),cyclo-(L-Tyr-L-Pro) (7),cyclo-(L-Hyp-L-Leu) (8),cyclo-(L-Leu-L-Ile) (9),cyclo-(L-Pro-L-Phe) (10),cyclo-(L-Val-L-Pro) (11),cyclo-(L-Pro-L-Leu) (12),cyclo-(L-Pro-L-Ile) (13),cyclo-(L-Val-L-Phe) (14),cyclo-(L-Val-L-Val) (15).All the compounds were isolated from S. prostrata for the first time.Moreover,compounds 2-4 and 7-15 were isolated from Selaginella genus for the first time.SH-SY5Y cell damage model induced by H₂O₂ was established by cell culture in vitro.The neuroprotective activity of the isolated compounds was evaluated by MTT assay.Compounds 5,6 and 8 were showed good neuroprotective activity at the concentration of 50 μmol/L.This study provided a theoretical basis for the research on the exploitation and utilization of S. prostrata.