A series of S-allyl-l-cysteine (SAC) with garlic acid conjugates as anti-inflammatory agents were designed and synthesized. Among the 40 tested compounds, SMU-8c exhibited the most potent inhibitory activity to Pam3CSK4-induced nitric oxide (NO) in RAW264.7 macrophages with IC50 of 22.54 ± 2.60 μM. The structure-activity relationship (SAR) study suggested that the esterified carboxyl group, carbon chain extension and methoxylation phenol hydroxy could improve the anti-inflammatory efficacy. Preliminary anti-inflammatory mechanism studies showed that SMU-8c significantly down-regulated the levels of Pam3CSK4 triggered TNF-α cytokine in human THP-1 cells, mouse RAW 264.7 macrophages, as well as in ex-vivo human peripheral blood mononuclear cells (PBMC) with no influence on cell viability. SMU-8c specifically blocked the Pam3CSK4 ignited secreted embryonic alkaline phosphatase (SEAP) signaling with no influence to Poly I:C or LPS triggered TLR3 or TLR4 signaling. Moreover, SMU-8c suppressed TLR2 in HEK-Blue hTLR2 cells and inhibited the formation of TLR1-TLR2, and TLR2-TLR6 complex in human PBMC. In summary, SMU-8c inhibited the TLR2 signaling pathway to down-regulate the inflammation cytokines, such as NO, SEAP and TNF-α, to realize its anti-inflammatory activity.