The function of IRS-1 is regulated by both tyrosine and serine/threonine phosphorylation. Phosphorylation of some serine/threonine residues in IRS-1 dampens insulin signaling, while phosphorylation of other serine/threonine residues enhances insulin signaling. Phosphorylation of Ser⁶²⁹ in human IRS-1 was identified through mass spectrometry analysis of IRS-1 isolated from human vastus lateralis muscle. Phosphorylation of Ser⁶²⁹ was increased by insulin in CHO/IR cells (1.26 ± 0.09 fold, P < 0.05) and L6 cells (1.35 ± 0.29 fold, P < 0.05) expressing human IRS-1. Sequence analysis surrounding Ser⁶²⁹ revealed conformity to the consensus phosphorylation sequence recognized by Akt. Phosphorylation of IRS-1 at Ser⁶²⁹ in cells was decreased upon treatment with either an Akt inhibitor or by co-expression with kinase dead Akt, while Ser⁶²⁹ phosphorylation was increased by co-expression with constitutively active Akt. In addition, Ser⁶²⁹ of IRS-1 is directly phosphorylated by Akt in vitro. In cells, preventing phosphorylation of Ser⁶²⁹ by means of a Ser⁶²⁹Ala mutation resulted in increased phosphorylation of Ser⁶³⁶, a known negative regulator of IRS-1, without affecting phosphorylation of Tyr⁶³² or Ser⁶¹⁶. Cells expressing the Ser⁶²⁹Ala mutation, along with increased Ser⁶³⁶ phosphorylation, had decreased insulin-stimulated association of the p85 regulatory subunit of PI 3-kinase with IRS-1 and decreased phosphorylation of Akt at Ser⁴⁷³. Finally, in vitro phosphorylation of a Ser⁶²⁹-containing IRS-1 fragment with Akt reduces the subsequent ability of Erk to phosphorylate Ser^636/639. These results suggest that a feed-forward mechanism may exist whereby insulin activation of Akt leads to phosphorylation of IRS-1 at Ser⁶²⁹, resulting in decreased phosphorylation of IRS-1 at Ser⁶³⁶ and enhanced downstream signaling. Understanding the complex phosphorylation patterns of IRS-1 is crucial to elucidating the factors contributing to insulin resistance and ultimately, the pathogenesis of type 2 diabetes.