Multisite phosphorylation of kinases can induce on-off or graded regulation of catalytic activity; however， its influence on substrate specificity remains unclear. Here， we show that multisite phosphorylation of ribosomal protein S6 kinase 1 (S6K1) alters target selection. Agonist-inducible phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) by S6K1 in monocytes and adipocytes requires not only canonical phosphorylation at Thr by mTORC1 but also phosphorylation at Ser and Ser in the C terminus by cyclin-dependent kinase 5 (Cdk5). S6K1 phosphorylation at these additional sites induces a conformational switch and is essential for high-affinity binding and phosphorylation of EPRS， but not canonical S6K1 targets， e.g.， ribosomal protein S6. Unbiased proteomic analysis identified additional targets phosphorylated by multisite phosphorylated S6K1 in insulin-stimulated adipocytes-namely， coenzyme A synthase， lipocalin 2， and cortactin. Thus， embedded within S6K1 is a target-selective kinase phospho-code that integrates signals from mTORC1 and Cdk5 to direct an insulin-stimulated， post-translational metabolon determining adipocyte lipid metabolism.