This study aims to investigate the protective effect of extract of Hypericum perforatum L. (HPE) on ulcerative colitis (UC) in mice. The chemical component of HPE was analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). And a mouse model of UC induced by dextran sulfate sodium (DSS) was established to evaluate the effects of HPE (300 mg/kg and 100 mg/kg) on the pathological changes of the colon tissue, as well as the expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and the impact on the gut microbiota in mice. The results of the chemical composition analysis showed that a total of 719 components were detected, among which fatty acids accounted for 20.03%; flavonoids accounted for 18.78%; terpenoids accounted for 16.55%; phenolic acids accounted for 8.62%; alkaloids accounted for 7.93%; polyketides accounted for 6.95%; phenylpropanoids and lignins accounted for 6.68%; amino acids and peptides accounted for 5.01%; coumarins accounted for 4.31%; carbohydrates accounted for 4.03%; and other compounds accounted for 1.11%. The relative content ratio of hyperin and hypericin, two characteristic compounds of H. perforatum, were 1.05% and 0.73% respectively. The results of the pharmacological experiments indicated that HPE significantly improved the indicators such as body weight loss, bloody stool, and shortened colon length in UC mice. The pathological features of UC, including destruction of colonic crypt structure, mucosal swelling, and inflammatory infiltration, were significantly improved by HPE. Also, the expression levels of IL-1β, IL-6 and TNF-α in the HPE administrated mice were significantly reduced. The 16S rDNA sequencing results showed that compared with the model group, the abundance of Dubosiella in the high-dose (300 mg/kg) and low-dose HPE groups (100 mg/kg) was significantly increased, while the abundances of Turicibacter and Cryptobacteroides were significantly decreased. In conclusion, the main chemical components of HPE mainly are flavonoids and phenylpropanoids. It can improve DSS-induced UC by modulating gut microbiota and inhibiting inflammation. The present study offers a valuable reference for the development and treatment of UC using H. perforatum.