Protein abundance tends to be more evolutionarily conserved than mRNA levels both within and between species, yet the mechanisms underlying this phenomenon remain largely unknown. Upstream open reading frames (uORFs) are widespreadcis-regulatory elements in eukaryotic genomes that regulate translation, but it remains unclear whether and how uORFs contribute to stabilizing protein levels. In this study, we performed ribosome translation simulations on mRNA to quantitatively assess the extent to which uORF translation influences the translational variability of downstream coding sequences (CDS) across varying contexts. Our simulations revealed that uORF translation dampens CDS translational variability, with buffering capacity increasing in proportion to uORF efficiency, length, and number. We then compared the translatomes at different developmental stages of twoDrosophilaspecies, demonstrating that uORFs buffer mRNA translation fluctuations during both evolution and development. Experimentally, deleting a uORF in thebcdgene—a prominent example of translational buffering—resulted in extensive changes in gene expression and phenotypes inDrosophila melanogaster. Additionally, we observed uORF-mediated buffering between primates and within human populations. Together, our results reveal a novel regulatory mechanism by which uORFs stabilize gene translation during development and across evolutionary time.