Understanding the determinants of the rate of protein sequence evolution is of fundamental importance in evolutionary biology. Many recent studies have focused on the yeast because of the availability of many genome-wide expressional and functional data. Yeast studies revealed a predominant role of gene expression level and a minor role of gene essentiality in determining the rate of protein sequence evolution. Whether these rules apply to complex organisms such as mammals is unclear. Here we assemble a list of 1,138 essential and 2,341 nonessential mouse genes based on targeted gene deletion experiments and report a significant impact of gene essentiality on the rate of mammalian protein evolution. Gene expression level has virtually no effect, although tissue specificity in expression pattern has a strong influence. Unexpectedly, gene compactness, measured by average intron size and untranslated region length, has the greatest influence. Hence, the relative importance of the various factors in determining the rate of mammalian protein evolution is gene compactness > gene essentiality approximately tissue specificity > expression level. Our results suggest a considerable variation in rate determinants between unicellular organisms such as the yeast and multicellular organisms such as mammals.