The roles of miR-9 in tumor progression are well-defined, however, its roles and the mechanisms in sepsis are still unclear. This work aims to explore miR-9 roles and related mechanism in LPS-induced sepsis. We found that miR-9 level was significantly upregulated in septic patients and RAW264.7 cells with LPS treatment, knockdown of miR-9 attenuated the induced effects of LPS on IL-6 and TNF-α secretion. In vivo experiments showed that miR-9 expression was increased in septic mice and knockdown of miR-9 partially reversed the upregulation of IL-6 and TNF-α levels in septic mice and prolonged the survival of septic mice. Mechanistic studies revealed that miR-9 could target ANO1, which inactivates the TGF-β/Smad2 signaling. Conversely, Smad2 could bind to the promoter of miR-9 and promote miR-9 expression. Notably, ANO1 overexpression or Smad2 knockdown attenuated miR-9 knockdown-mediated inhibition on LPS-induced sepsis. Therefore, these results suggest that the negative Smad2/miR-9/ANO1 regulatory loop is responsible for LPS-induced sepsis.