Background <p>Isothiocyanate sulforaphane (SFN), found in cruciferous vegetables, is known for its anti-inflammatory properties in vitro, in vivo, and in humans. After intestinal absorption, SFN forms metabolites such as SFN-glutathione, SFN-cysteine, and SFN-N-acetylcysteine in plasma and tissues, which may contribute to its anti-inflammatory effects. This study aimed to compare the anti-inflammatory profiles of these metabolites with that of SFN, focusing on proinflammatory pathways such as TLR4/NF-κB and NLRP3 inflammasome.</p> Methods <p>The study utilized human TLR4 Reporter HEK293 cells and human THP-1 macrophages to investigate the pro-inflammatory effects of lipopolysaccharide (LPS). TLR4 activation was assessed in the HEK293 cells, while the activation of NF-κB p65, NLRP3, and Keap1/Nrf2, as well as the release of the pro-inflammatory cytokines IL-1β and TNF-α, were evaluated in THP-1 macrophages through western blotting, RT-PCR, and ELISA techniques.</p> Results <p>SFN and its metabolites decreased the activation of TLR4, NLRP3 inflammasome, and nuclear NF-κB p65. Further, they reduced the expression and release of the cytokines IL-1β and TNF-α. While all compounds disrupted the Keap1/Nrf2 complex and released individual proteins, only SFN promoted the nuclear translocation of Nrf2, suggesting a different crosstalk with the NF-κB pathway.</p> Conclusions <p>SFN metabolites, including SFN-glutathione, SFN-cysteine, and SFN-N-acetylcysteine, have demonstrated anti-inflammatory effects mainly through the TLR4/NF-κB pathway, which involves the NLRP3 inflammasome. This data indicates their direct contribution to the overall anti-inflammatory effects of SFN.</p>