Several members of the Schlafen (SLFN) protein family exhibit antiviral activity. The antiviral effects of SLFN11, SLFN13, and SLFN14 correlate with their capacity to inhibit translation of viral mRNAs enriched in rare codons, a process that requires their intrinsic endoribonuclease activity. SLFN11 and SLFN13 restrict viral replication by degrading tRNAs, thereby reducing the availability of these tRNAs below a critical threshold necessary for efficient translation of rare-codon–biased viral transcripts. In contrast, the molecular mechanism underlying the antiviral function of SLFN14 remains poorly defined. Previous studies have shown that SLFN14 does not selectively degrade rare-codon–rich mRNAs, suggesting that it may target ribosomal or transfer RNAs within ribosomes engaged in viral mRNA translation. To further elucidate the mechanism of SLFN14-mediated antiviral activity, we mapped the protein regions required for its function. Analysis of a panel of SLFN14 deletion mutants demonstrated that a C-terminally truncated variant (residues 1–373) retained both ribosome-binding and antiviral activity. Furthermore, a mutant lacking both the C-terminal region and an additional N-terminal segment (residues 121–328) remained capable of ribosome association and inhibition of HIV-1 replication. These findings indicate that the region encompassing the catalytic residues (clustered between residues 206 and 249), together with the ribosome-binding domain, is sufficient to mediate the antiviral activity of SLFN14.