Huangqin Decoction (HQD), a traditional Chinese medicine formula from the Shang Han Lun written by Zhang Zhongjing, has been used in China for nearly two thousand years. According to the traditional Chinese medicine and previous literature, HQD has the effect of clearing heat, removing toxins, relieving diarrhea and pain. Therefore, HQD was used to prevent or cure many diseases, such as inflammation, diarrhea, malaria, and other acute or chronic gastrointestinal diseases. The effect of HQD, one-herb-absent HQD treatments and enrofloxacin (ENR) on the average daily gain (ADG), mortality rates, visceral index and toll-like receptors (TLRs), inflammatory factors and intestinal microflora in E. coli O78-inoculated chicks were investigated. HQD supplementation increased ADG and reduced the mortality rates caused by E. coli challenge, decreased the heart, liver, bursa of Fabricius (BF) and spleen index. HQD supplementation decreased the serum lysozyme (LZM), IL-1β, TNF-α, IL-10, IL-6 level, down-regulated the mRNA expression of TLR4, -5 and -15 in the spleen by E. coli challenged chicks, and up-regulated the mRNA expression of TLR4, -5 and -15 in BF. At the phylum level, HQD supplementation reversed the increase of Operational Taxonomic Unit (OTUs), decreased the relative abundance of harmful bacteria Proteobacteria, increased the relative abundance of probiotic bacteria Bacteroidetes and Firmicutes. At the genus level, HQD decreased the relative abundance of harmful bacteria Escherichia-Shigella and Pseudomonas. It means that HQD treatment reversed the change of the gut microbiota structure. Compared with HQD, HQD-DZ and HQD-HQ increased the mortality rates. HQD-HQ decreased the ADG and liver index. HQD-GC decreased the spleen index. All herb-absent increased the serum IL-6, but only the HQD-HQ and HQD-SY increased the serum TNF-α. All herb-absent did not activate the TLRs signaling pathways in spleen and BF of chicks. The harmful bacteria Escherichia-Shigella were increased in HQD-HQ and HQD-DZ treatments. HQD-DZ treatment also increased the level of Proteobacteria. The results showed that dietary supplementation with HQD, by down-regulating the mRNA expression of TLR4, -5 and -15 in the spleen, further decreasing the serum LZM and IL-1β, TNF-α, IL-10, IL-6 level, improves the immune function and reverses the change of fecal microbiome in chicks challenged with E. coli. In herb-absent supplementation, the results showed that SY and DZ play a key role in reducing the levels of inflammatory factors and keeping fecal microbiome balance respectively. More importantly, HQ is indispensable in HQD, not only play a key role in reducing the level of inflammatory factors, but also in keeping the balance of fecal microflora.