To investigate the anti-inflammatory effect and mechanism of proper concentration of short-chain fatty acids (SCFAs) mixture (SCFAs mix) on microglia under inflammatory environment. In this study, the murine microglial cell line BV-2 treated with lipopolysaccharide (LPS) was used as an inflammatory cell model. BV-2 cells were treated with different concentrations of sodium acetate, sodium propionate and sodium butyrate, and SCFAs mix, the cell viability were detected by CCK8 kit. The concentrations of each SCFA and SCFAs mixture (SCFAs mix) will be selected by double standard: having no effect on cell viability and having the anti-inflammation effect. The examination of anti-inflammation effect and its mechanism of SCFAs mix in proper concentration on LPS-stimulated BV-2 cells include: (a) the production of NO of BV-2 cells was detected by NO kit, (b) the inflammatory factors such as TNF-α and IL-6 released into the culture supernatant by BV-2 cells were detected by ELISA, (c) the expression of inflammatory factors such as TNF-α and IL-6, inflammasome NLRP3 and some key factors within inflammatory pathways such as TLR4 and NF-κB, were detected by qRT-PCR and Western-blot. We found that after 4 hours of LPS stimulation on BV-2 cells, the addition of a certain concentration of single SCFA to the system for 12 h could not alleviate the inflammatory response of BV-2 cells, but SCFAs mix with the same terminal concentration of each SCFAs could significantly reduce the levels of NO (P<0.001), TNF-α (P<0.001) and IL-6 (P<0.001) in cell culture supernatants. Simultaneously, SCFAs mix inhibited the increase of iNOS, TNF-α, IL-6 (P<0.001) and inflammasome NLRP3 (P<0.001) mRNA in BV-2 cells induced by LPS. Further study showed that SCFAs mix could inhibit LPS-induced high expression of TLR4, MyD88, TRAF6 and NF-κB proteins, which were the key molecules of the inflammatory signaling pathway in BV-2 cells. In conclusion, proper concentration of SCFAs mix could function as a protective role to inhibit LPS-induced microglial inflammatory responses by regulation of the TLR4/MyD88/TRAF6/NF-κB signaling pathway.