Thioredoxin (TRX) is generally a 12-kDa protein-disulfide reductase. Here, we report the discovery of a 16-kDa thioredoxin-related protein designated Cr-TRP16, from a "living fossil," the horseshoe crab (Carcinoscorpius rotundicauda). Cr-TRP16 contains an atypical WCPPC catalytic motif and possesses the classical thiodisulfide reductase activity, as indicated by the insulin reduction assay. Furthermore, Cr-TRP16 can function as an antioxidant and protect against DNA nicking by reactive oxygen species. Overexpression of Cr-TRP16 regulated the transcription of NF-kappaB-dependent genes probably by enhancing NF-kappaB DNA-binding activity, suggesting possible roles for Cr-TRP16 in modulating the NF-kappaB signaling pathway. In vivo, the antioxidant pyrrolidine dithiocarbamate suppressed the expression of NF-kappaB-regulated genes such as IkappaB and inducible nitric oxide synthase. This further supports the notion that oxidative stress is also a regulatory factor of the NF-kappaB signaling pathway, a phenomenon which has been entrenched for several hundred million years. Furthermore, we demonstrated that the 16-kDa thioredoxins are evolutionarily conserved from Caenorhabditis elegans to human. Interestingly, thioredoxin-like 6, a human homologue of Cr-TRP16, could also enhance NF-kappaB DNA-binding activity, suggesting that the regulatory role of the 16-kDa thioredoxins on NF-kappaB is well conserved through evolution.