Background: Skin squamous cell carcinoma (SSCC) is a common malignancy of skin, and its occurrence is a multifactorial process. Glycolysis plays a vital role in its occurrence and development. This study investigated the effect of neurogenic locus notch homolog protein 1 (Notch1) on malignant behavior of SSCC cells through glycolysis mediated by hypoxia-inducible factor 1 alpha (HIF1A).Methods: Notch1 expressions in normal human skin cells human keratinocytes (HaCaT) and three SSCC cell lines (SCL-1, Colo-16, and A431) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Colo-16 cells with Notch1 or HIF1A overexpression were constructed by liposome-mediated transfection technology. Colo-16 cells were divided into negative control of Notch1 overexpression (pc-NC1), Notch1 overexpression (pc-Notch1), Notch1 overexpression+negative control of HIF1A overexpression (pc-Notch1+pc-NC2), and Notch1 overexpression+HIF1A overexpression (pc-Notch1+pc-HIF1A) groups. Clone formation, scratch healing and Transwell tests were used to detect the effect of Notch1 overexpression on Colo-16 cell proliferation, migration and invasion ability, respectively. Glucose and lactic acid test kits were used to detect glucose and lactic acid levels. The protein expression levels of Notch1, HIF1A and key glycolytic proteases were detected by western blot.Results: Notch1 expression in SSCC cells was lower than in the control group (p < 0.01, p < 0.001). Notch1 overexpression reduced cell proliferation, invasion and migration, and inhibited glucose consumption and lactic acid formation (p < 0.01, p < 0.001). Notch1 overexpression also inhibited expression of glucose transporter type 1 (GLUT1) and related key glycolytic proteases (p < 0.01, p < 0.001, p < 0.0001). Upregulation of Notch1 decreased HIF1A protein expression (p < 0.01, p <0.001). HIF1A overexpression partially reversed the inhibitory effect of Notch1 on cell malignant behavior and glycolysis. Conclusions: Notch1 inhibits proliferation, migration and invasion of SSCC cells through HIF1A-mediated glycolysis and may be a target for SSCC diagnosis and treatment.