RNA binding proteins (RBPs) can regulate the stability, localization, and translation of their target mRNAs. Among them, Puf3p is a well-known Pumilio family RBP whose biology has been intensively studied. Nevertheless, the impact of Puf3p on the translational regulation of its downstream genes still remains to be investigated at the genome-wide level. In this study, we combined ribosome profiling and RNA-Seq in budding yeast (Saccharomyces cerevisiae) to investigate Puf3p's functions in translational regulation. Comparison of translational efficiency (TE) between wild-type and puf3Δ strains demonstrates extensive translational modulation in the absence of Puf3p (over 27% genes are affected at the genome level). Besides confirming its known role in regulating mitochondrial metabolism, our data demonstrate that Puf3p serves as a key post-transcriptional regulator of downstream RBPs by regulating their translational efficiencies, indicating a network of interactions among RBPs at the post-transcriptional level. Furthermore, Puf3p switches the balance of translational flux between mitochondrial and cytosolic ribosome biogenesis to adapt to changes in cellular metabolism. In summary, our results indicate that TE can be utilized as an informative index to interrogate the mechanism underlying RBP functions, and provide novel insights into Puf3p's mode-of-action.