Purpose: Berberine (BBR), a major ingredient extracted from Coptis chinensis, is a natural drug with limited oral bioavailability. We developed nanostructured lipid carriers (NLCs) as a delivery system for enhanced anti-inflammatory activity of BBR against ulcerative colitis (UC). Methods: BBR-loaded nanostructured lipid carriers (BBR-NLCs) prepared via high-pressure homogenization were evaluated for particle size, zeta potential, drug entrapment efficiency, drug loading, drug release, toxicity, and cellular uptake. The anti-UC activities of free and encapsulated BBR were evaluated in a DSS-induced acute model of UC in mice. Results: Spherical BBR-NLCs were prepared with a particle size of 63.96± 0.31 nm, a zeta potential of +3.16 ± 0.05 mV, an entrapment efficiency of 101.97± 6.34%, and a drug loading of 6.00± 0.09%. BBR-NLCs showed excellent biocompatibility in vivo. Cellular uptake experiments showed that BBR-NLCs improved uptake of BBR by RAW 264.7 cells and Caco-2 cells. Oral administration of BBR-NLCs significantly alleviated colitis symptoms (DAI, colon length, spleen swelling, MPO activity) through inhibition of NF-κB nuclear translocation, decreased expression of pro-inflammatory cytokines (IL-1β, IL-6, MMP-9, CX3CR1, COX-2, TERT), and increased expression of the tight junction protein ZO-1. Conclusion: BBR-loaded NLCs improved colitis symptoms, which suggested that this may be a novel formulation for treatment of UC.