The purpose of this study was to investigate the anti-inflammatory effects of Perilla Seed Extract (PSE) and its active ingredient on Inflammatory Bowel Disease (IBD) in vitro and in vivo. Thirty-two C57/BL mice were randomly divided into four groups ( = 8): control group (CON), PBS group, LPS group (LPS 3.5 mg/kg given intraperitoneally [ip] on day 7 of the study only), and PSE group (100 mg/kg orally daily + LPS ip at 3.5 mg/kg on day 7). Mice were euthanized 24 h after LPS administration. MODE-K cells were divided into five groups: control group (CON), LPS group (50 μg/mL LPS for 2 h), and PSE group (low dose, 25 μg/mL PSE + LPS; middle dose, 50 μg/mL PSE + LPS; high dose, 100 μg/mL PSE + LPS). In vivo, compared with the CON group, LPS revealed a significant decrease in the villus length-to-crypt depth ratio ( < 0.01) and goblet cell density per unit area ( < 0.01). Conversely, PSE administration resulted in a significant increase in the villus length-to-crypt depth ratio ( < 0.01) and goblet cell density ( < 0.01). LPS significantly increased the ROS content ( < 0.01), the secretion of inflammatory cytokines of IL-6 ( < 0.01), TNF-α ( < 0.01), and the mRNA expressions of ( < 0.01). LPS significantly decreased the mRNA expressions of ( < 0.01) and ( < 0.01). In contrast, PSE treatment led to a marked decrease in ROS levels ( < 0.01), along with a reduction in the secretion of inflammatory factors IL-6 ( < 0.01) and TNF-α( < 0.05), as well as the mRNA expressions of ( < 0.01). Concurrently, PSE significantly increased the mRNA expressions of ( < 0.05) and ( < 0.01). In vitro, PSE treatment also significantly reversed LPS-induced inflammation, oxidation and tight junction-related factors. Network pharmacology identified 97 potential targets for PSE in treating IBD, while transcriptomics analysis revealed 342 differentially expressed genes (DEGs). Network pharmacology and transcriptomics analysis indicated that significant pathways included the PI3K-Akt signaling pathway, MAPK signaling pathway, and TNF signaling pathway, of which the PI3K-AKT pathway may represent the primary mechanism. In an in vivo setting, compared with the CON group, LPS led to a significant increase in the protein expression of p-PI3K/PI3K ( < 0.01) and p-AKT1/AKT1 ( < 0.01). Conversely, PSE resulted in a significant decrease in the protein expression of p-PI3K/PI3K ( < 0.01) and p-AKT1/AKT1 ( < 0.01). In vitro, compared with the LPS group, PSE also significantly blocked the protein expression of p-PI3K/PI3K ( < 0.01) and p-AKT1/AKT1 ( < 0.01). The chemical composition of PSE was analyzed using UPLC-MS/MS, which identified six components including luteolin (content 0.41%), rosmarinic acid (content 0.27%), α-linolenic acid (content 1.2%), and oleic acid (content 0.2%). Molecular docking found that luteolin could establish stable binding with eight targets, and luteolin significantly decreased the p-AKT1/AKT1 ratio ( < 0.01) compared to the LPS group in MODE-K cells. In summary, PSE demonstrates efficacy against IBD progression by enhancing intestinal barrier function and inhibiting inflammatory responses and oxidative stress via the PI3K/AKT signaling pathway, and luteolin's inhibition of AKT1 protein phosphorylation appears to play a particularly crucial role in this therapeutic mechanism.