The RNA-binding protein Sex-lethal (Sxl) is an important post-transcriptional regulator of sex determination and dosage compensation in female Drosophila. To prevent the assembly of the MSL dosage compensation complex in female flies, Sxl acts as a repressor of male-specific lethal-2 (msl-2) mRNA translation. It uses two distinct and mutually reinforcing blocks to translation that operate on the 5 ′ ′ ′ ′ ′ and 3 ′ ′ ′ ′ ′ untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5 ′ ′ ′ ′ ′ UTR-mediated translational control involves an upstream open reading frame, 3 ′ ′ ′ ′ ′ UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr), which is recruited to the transcript by Sxl. We have identified the protein Sister-of-Sex-lethal (Ssx) as a novel repressor of translation with Sxl-like activity. Both proteins have a comparable RNA-binding specificity and can associate with uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5 ′ ′ ′ ′ ′ UTR of msl-2. However, Ssx is inactive in 3 ′ ′ ′ ′ ′ UTR-mediated regulation, as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain of function and repression via the 3 ′ ′ ′ ′ ′ UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway.