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Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems., Author summary Protein kinases, which catalyze the transfer of phosphate from ATP to substrate proteins, are important enzymes in cellular signal transduction pathways mediating responses to extracellular cues. In order to function properly in signal transmission, each kinase must phosphorylate only a limited number of proteins among the thousands present within the cell. One way that kinases choose their substrates is by recognition of amino-acid sequence motifs surrounding the site of phosphorylation, but kinases can also recruit substrates through docking interactions occurring outside of the catalytic cleft. For most kinases, the relative contribution of catalytic site and docking site interactions to substrate selection is not known. Here, we investigated the phosphorylation site specificity of a group of 30 human protein serine–threonine kinases called the STE20 family. Guided by x-ray crystal structures of kinase-peptide complexes, we re-engineered the catalytic clefts of two members of this group, PAK4 and MST4, to exchange their phosphorylation site motifs. In cells, the re-engineered form of PAK4 was unable to phosphorylate substrates of wild-type PAK4 or to carry out its normal function in reorganizing the actin cytoskeleton. In contrast, it was able to function in place of MST4 and related kinases in the growth-controlling Hippo signaling pathway. Overall, these studies suggest that catalytic site interactions can be both necessary and sufficient for kinase function.