Flaxseed linusorbs (FLs), cyclic peptides derived from flaxseed oils, have shown multiple activities such as anticancer, antibacterial, and anti-inflammatory effects. However, the anti-inflammatory monomers of FLs and their mechanisms are still unclear. In this study, we have elucidated that FLs suppress the modulation of NF-κB/MAPK signaling pathways by targeting the inhibition of activating TLR4 in LPS-induced RAW 264.7 cells. Therefore, the transcription and expression of inflammatory cytokines (i.e., TNF-α, IL-1β, and IL-6) and inflammatory mediator proteins (i.e., iNos and Cox-2) were significantly suppressed by FLs. In addition, an in silico study discovered that eight monomers of FLs showed high-affinity bindings with TLR4. In silico data combined with HPLC results indicated that FLA and FLE, accounting for 44%, were likely the major anti-inflammatory monomers in FLs. In summary, FLA and FLE were proposed as the main anti-inflammatory active cyclopeptides via hindering TLR4/NF-κB/MAPK signaling pathways, suggesting the potential use of food-derived FLs as natural anti-inflammatory supplements in a daily diet.