Lactic acid bacteria (LAB) can support host health and crosstalks between ligands on bacterial cells and Toll-like receptors (TLR) are known as key inducers of these health effects. LAB-secreted bioactive factors also possess health-promoting properties but the underlying mechanisms remain to be identified. Here, we report that secreted factors of different LAB strains induce cytokine (IL-10 and TNF-α) production by THP1-differentiated human macrophages in a species- and strain-dependent manner. LAB-secreted products also drive immune-stimulating and anti-pathogenic M1-macrophage polarization. Moreover, bacterial supernatants induce species- and strain-dependent TLR2 activation. Several bacterial strains such as L. casei CCFM9 and L. brevis CCFM498 cannot activate TLR signaling, but their secreted products strongly activate TLR2. This indicates that LAB-secreted components may further boost the functional effects of bacterial strains. Our results confirm direct interactions of LAB-secreted components with TLRs, and provide novel insights in how LABs may provide immune-modulation.