The role of S-layer proteins (SLP), which form the outermost layer of cell walls in lactic acid bacteria (LAB), plays a crucial role in regulating immune-stimulating activity, thereby closely influencing LAB’s ability to boost host immunity. In this study, the heterologous expression, purification, and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated. Initially, the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid. SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions, which was further demonstrated by western blot (WB) and LC-MS/MS. ELISA, WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors (TNF-α, IL-6, NO) and ROS via MAPK and NF-κB signaling pathways. The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.