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Abstract We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoanCapsaspora owczarzaki(Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allowsCo-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing ofCo-NF-κB is not induced by IκB kinases in human cells. OverexpressedCo-NF-κB localizes to the cytoplasm inCocells.Co-NF-κB mRNA and DNA-binding levels differ across threeCapsasporalife stages. RNA-sequencing and GO analyses identify possible gene targets ofCo-NF-κB. Three NF-κB-like proteins from the choanoflagellateAcanthoeca spectabilis(As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All threeAs-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.