Baicalin and berberine hydrochloride are the main chemical compositions of and respectively. and are two traditional Chinese herbs that are commonly used together in compounded formulations to treat colitis. Therefore, the combination of Baicalin and berberine hydrochloride (BBH) to treat colitis was studied. The results of pharmacological evaluations demonstrated the excellent protective effects of BBH on colitis induced by dextran sulfate sodium (DSS). BBH could improve the morphological condition of colitis in mice and maintain the balance of proinflammation cytokines (IL-6, IL-8, IL-1, and TNF-) and anti-inflammation cytokines (IL-4 and IL-10). The 16s rDNA sequencing revealed that BBH was able to modulate the composition of intestinal microflora, especially the abundances of , , , , The results of PICRUSt indicated that the therapeutic effects of BBH were tightly connected with DNA synthesis, replication and repair of gut microbiota. In summary, it was concluded that BBH could protect mice against DSS-induced colitis, and the protective effects were tightly correlated with gut microbiota.