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Lactic acid bacteria (LAB) are demonstrably beneficial on the digestive and immune systems. S-layer protein (SlpA), as the outermost component of the cell wall, exerts an anti-inflammatory activity in lactobacilli, while the pro-inflammatory properties of SlpA in cross-talk with immune cells have rarely been investigated. Here, we showed that SlpA isolated from Lactobacillus acidophilus CICC 6074 exhibits more pronounced pro-inflammatory behavior through activating MAPK and NF-κB signaling, which was further corroborated by an increase in the NF-κB translocation from cytoplasm to the nucleus and DNA binding activity of NF-κB, triggered by SlpA treatment. Notably, a specific PKC inhibitor, chelerythrine, significantly blocked SlpA-elicited pro-inflammatory response, indicating that SlpA could act as stimulators of the innate immune system by triggering PKC to MAPK and NF-κB signaling pathway. This study provides important insights on the immunomodulatory properties of cell surface components in lactobacilli.