Toll-like receptor 2 (TLR2) responses are involved in various inflammatory immune disorders. Phloretin is a naturally occurring dietary flavonoid that is abundant in fruit. Here, we investigated whether the anti-inflammatory activity of phloretin is mediated through TLR2 pathways, and whether phloretin acts as an inhibitor of TLR2/1 heterodimerization using the TLR2/1 agonist Pam₃CSK₄. We tested the effects of phloretin on tumor necrosis factor (TNF)-α production induced by various TLRs using known TLR-specific agonists. Phloretin significantly inhibited Pam₃CSK₄-induced TRL2/1 signaling in Raw264.7 cells compared to TLR signaling induced by the other agonists tested. Therefore, we further tested the effects of phloretin in human embryonic kidney (HEK) 293-hTLR2 cells induced by Pam₃CSK₄, and confirmed that phloretin has comparable inhibition of TLR2/1 heterodimerization to that induced by the known TLR2 inhibitor CU-CPT22. Moreover, phloretin reduced the secretion of the inflammatory cytokines TNF-α and interleukin (IL)-8 in Pam₃CSK₄-induced HEK293-hTLR2 cells, whereas it did not significantly reduce these cytokines under Pam₂CSK₄-induced activation. Western blot results showed that phloretin significantly suppressed Pam₃CSK₄-induced TLR2 and NF-κB p65 expression. The molecular interactions between phloretin and TLR2 were investigated using bio-layer interferometry and in silico docking. Phloretin bound to TLR2 with micromolar binding affinity, and we proposed a binding model of phloretin at the TLR2⁻TLR1 interface. Overall, we confirmed that phloretin inhibits the heterodimerization of TLR2/1, highlighting TLR2 signaling as a therapeutic target for treating TLR2-mediated inflammatory immune diseases.