Schlafen proteins are important in cell differentiation and defense against viruses, and yet this family of vertebrate proteins is just beginning to be understood at the molecular level. Here, the three-dimensional architecture and molecular interfaces of human schlafen12 (hSLFN12), which promotes intestinal stem cell differentiation, are analyzed by sequence conservation and structural modeling in light of the functions of its homologs and binding partners. Our analysis shows that the schlafen or divergent AAA ATPase domain described in the N-terminal region of schlafens in databases and the literature is a misannotation. This N-terminal region is conclusively an AlbA_2 DNA/RNA binding domain, forming the conserved core of schlafens and their sequence homologs from bacteria through mammals. Group III schlafens additionally contain a AAA NTPase domain in their C-terminal helicase region. In hSLFN12, we have uncovered a domain matching rho GTPases, which directly follows the AlbA_2 domain in all group II-III schlafens. Potential roles for the GTPase-like domain include antiviral activity and cytoskeletal interactions that contribute to nucleocytoplasmic shuttling and cell polarization during differentiation. Based on features conserved with rSlfn13, the AlbA_2 region in hSLFN12 is likely to bind RNA, possibly as a ribonuclease. We hypothesize that RNA binding by hSLFN12 contributes to an RNA-induced transcriptional silencing/E3 ligase complex, given the functions of hSLFN12's partners, SUV39H1, JMJD6, and PDLIM7. hSLFN12's partner hSerpinB12 may contribute to heterochromatin formation, based on its homology to MENT, or directly regulate transcription via its binding to RNA polymerase II. The analysis presented here provides clear architectural and transcriptional regulation hypotheses to guide experimental design for hSLFN12 and the thousands of schlafens that share its motifs.