Ulcerative colitis (UC) is a chronic inflammatory bowel disease underpinned by barrier dysfunction, oxidative stress, and persistent immune activation. This study evaluated the therapeutic efficacy and mechanistic basis of an ethanol extract from Pingyin rose petals (PRE) in mice with dextran sulfate sodium (DSS)-induced colitis. Animals received PRE (50 or 100 mg·kg?¹) throughout 3% DSS exposure. PRE markedly reduced disease activity and body weight loss. Transcriptome analysis revealed restored tight junction protein expression (Zo-1, +65.34%; Occludin, +72.10%; Claudin-1, +58.45%), while biochemical assays showed enhanced antioxidant capacity (SOD elevated by 248% in serum and 46% in colon; MDA reduced by 36% and 57%) and downregulated TLR4/NF-κB pathway-associated genes at the transcriptional level (Tlr4, ?64.85%; Tnf-α, ?86.00%; Il-1β, ?86.30%). Metabolically, PRE restored cecal short-chain fatty acids in a dose-dependent manner, with recovery of the six major SCFAs exceeding 55% and 95% at the low (50 mg·kg-¹) and high (100 mg·kg-¹) doses, respectively. Collectively, these results establish that PRE, as a multicomponent botanical extract, confers anti-colitic protection through the integrated modulation of epithelial barrier integrity, oxidative homeostasis, and transcriptional downregulation of TLR4/NF-κB pathway-related genes.