Notch signaling is controlled at multiple levels. In particular, stabilized Notch receptor activation directly affects the transcriptional activations of Notch target genes. Although some progress has been made in terms of defining the regulatory mechanism that alters Notch stability, it has not been determined whether Notch1/NICD stability is regulated by GSK-3alpha. Here, we show that Notch1/NICD levels are significantly regulated by GSK-3beta and by GSK-3alpha. Treatment with LiCl (a specific GSK-3 inhibitor) or the overexpression of the kinase-inactive forms of GSK-3alpha/beta significantly increased Notch1/NICD levels. Endogenous NICD levels were also increased by either GSK-3alpha/beta- or GSK-3alpha-specific siRNA. Furthermore, it was found that GSK-3alpha binds to Notch1. Deletion analysis showed that at least three Thr residues in Notch1 (Thr-1851, 2123, and 2125) are critical for its response to LiCl, which increased not only the transcriptional activity of endogenous NICD but also Hes1 mRNA levels. Taken together, our results indicate that GSK-3alpha is a negative regulator of Notch1/NICD.