The testis brain RNA-binding protein (TB-RBP/translin) is a DNA- and RNA-binding protein with multiple functions. As an RNA-binding protein, TB-RBP binds to conserved sequence elements often present in the 3′ untranslated regions (UTRs) of specific mRNAs modulating their translation and transport. To identify additional mRNA targets of TB-RBP, immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) assays were carried out using an affinity-purified antibody to TB-RBP with testicular extracts. Gapds mRNA was found to be selectively precipitated in a TB-RBP-mRNA complex. Consistent with the delayed translation of GAPDS and the subcellular ribonucleoprotein location of TB-RBP, polysomal gradient analysis showed that most of the Gapds mRNA in adult testis extracts was present in the nonpolysomal fractions. In vitro translation assays revealed that Gapds mRNA translation was inhibited by recombinant TB-RBP or by a TB-RBP mutant protein, Nb, capable of binding RNA. No inhibition was seen with mutant forms of TB-RBP lacking domains required for RNA binding, including the TB-RBP Cb mutant and the C-terminal-truncated form of TB-RBP that disrupts the leucine zipper. As an additional indicator of the specificity of TB-RBP inhibition of Gapds mRNA translation, a putative TB-RBP binding H-element was deleted from the 5′ UTR of the Gapds mRNA. No translational inhibition by recombinant TB-RBP was seen with Gapds mRNA lacking the H element. These data suggest that TB-RBP is involved in the posttranscriptional regulation of Gapds gene expression during spermiogenesis. Moreover, the Gapds mRNA is the first mRNA shown to have a functional TB-RBP binding site in its 5′ UTR.