Initially discovered in Drosophila, the Hippo pathway is pivotal for tissue growth and organ homeostasis. Regulated by both extrinsic and intrinsic signals, the Hippo pathway exerts its effect via a core kinase cascade, in which Large tumor suppressor 1 and 2 (LATS1/2) plays a key role. LATS1 has been shown to regulate mitotic progression by phosphorylate myosin phosphatase target subunit 1 (MYPT1) to counteract polo-like kinase (PLK1) activity, a mitotic master kinase. Herein we demonstrate that the hexosamine biosynthetic pathway regulates the Hippo pathway via LATS1. We show that LATS1 interacts with the O-GlcNAc transferase (OGT) and is O-GlcNAcylated. Via electron transfer dissociation mass spectrometry, we mapped the O-GlcNAcylation sites to be Ser479/Ser482/Thr484/Thr485. O-GlcNAcylation attenuates LATS1 protein stability, and downregulates the phosphorylation level of its downstream substrates, such MYPT1. Subsequently, decreased MYPT1-pS473 levels enhanced PLK1-pT210 levels and drives mitosis. Importantly, we demonstrate that in Drosophila O-GlcNAcylation of LATS1 promotes the fly wing size. Thus, this study suggests that O-GlcNAcylation links extrinsic glucose levels to LAST1 in the Hippo pathway and cell proliferation.