Salmonella entericaserovar typhimurium (S.Typhimurium) is a common Gram-negative foodborne pathogenic bacterium that causes gastrointestinal disease in humans and animals. It is well known that adhesins and invasins play crucial roles in the infection mechanism ofS.Typhimurium.S.Typhimurium STM0306 has been denoted as a putative protein and its functions have rarely been reported. In this study, we constructed theSTM0306gene mutant strain ofS.Typhimurium and purified the recombinant STM0306 fromEscherichia coli. Deletion of theSTM0306gene resulted in reduced adhesion and invasion ofS.Typhimurium to IPEC-J2, Caco-2, and RAW264.7 cells. In addition, STM0306 could bind to intestinal epithelial cells and induced F-actin modulation in IPEC-J2 cells. Furthermore, we found that STM0306 activated the nuclear factor kappa B (NF-κB) signaling pathway and increased the mRNA expression of pro-inflammatory cytokines such as IL-1β, TNF-α, as well as chemokine CXCL2, thus resulting in cellular inflammation in host cells. In vivo, the deletion of theSTM0306gene led to reduced pathogenicity ofS.Typhimurium, as evidenced by lower fecal bacterial counts and reduced body weight loss inS.Typhimurium infected mice. In conclusion, the STM0306 ofS.Typhimurium is an important adhesin/invasin involved in the pathogenic process and cellular inflammation of the host.