Oral nanoparticles have been considered a prospective drug delivery carrier against ulcerative colitis (UC). To enhance the mucus-penetrating capacity and aqueous solubility, and strengthen the anti-inflammatory effect of resveratrol (RSV), we fabricated RSV-loaded silk fibroin-based nanoparticles with the functionalization of Pluronic F127 (PF-127). The obtained PF-127-functionalized RSV-loaded NPs had an average particle size around 170 nm, a narrow size distribution (polydispersity index < 0.2), and negative zeta potential (−20.5 mV). Our results indicated that the introduction of PF-127 strengthened the mucus-penetrating property of NPs. In vitro studies suggested that NPs with PF-127 enhanced the suppression of the secretion of proinflammatory cytokine TNF-α and reactive oxygen species (ROS) from RAW 264.7 macrophages under lipopolysaccharide stimulation in comparison with other counterparts. According to the evaluation of macro symptoms and main inflammatory cytokines, we further report preferable therapeutic outcomes achieved by PF-127 functionalized-NP-treated dextran sulphate sodium (DSS) groups in the colitis model compared with blank silk fibroin NPs and RSV-loaded NPs without the functionalization of PF-127. Taken together, this work suggests that the fabricated PF-127 NPs via the oral route are promising and useful RSV-loaded nanocarriers for UC treatment.