The introduction of multiple genes into cells is increasingly required for understanding and engineering biological systems. Small-molecule–responsive transcriptional regulation has been widely used to control transgene expression. In contrast, methods for specific and simultaneous regulation of multiple genes with a single regulatory protein remain undeveloped. In this report, we describe a method for quantitatively tuning the expression of multiple transgenes with a translational regulatory protein. A protein that binds a specific RNA motif inserted in the 5′-untranslated region (UTR) of an mRNA modulates the translation of that message in mammalian cells. We provide two independent mechanisms by which to rationally fine-tune the output: the efficiency of translation correlates well with the distance between the inserted motif and the 5′ terminus of the mRNA and is further modulated by the tandem insertion of multiple RNA motifs. The combination of these two approaches allowed us to fine-tune the translational efficiency of target mRNAs over a wide dynamic range. Moreover, we controlled the expression of two transgenes simultaneously and specifically by engineering eachcis-regulatory 5′-UTR. The approach provides a useful alternative regulatory layer for controlling gene expression in biological research and engineering.