Objective To investigate the role of STK4?AS1 in regulating the proliferation， invasion， and migration of esophageal squamous cell carcinoma （ESCC） cells through the MYG1/Notch signaling pathway. Methods Quantitative real?time PCR （qRT?PCR） was used to detect the expression of STK4?AS1 in ESCC cells. MTS assay， wound healing and Transwell assay were conducted to explore the proliferation， migration， and invasion abilities in each group in Eca109 and Kyse150 cells. mRNA sequencing （mRNA?seq） was used to detect the downstream target genes of STK4?AS1. KEGG functional enrichment analyses were used to predict the possible biological processes and signaling pathways. qRT?PCR and western blot were performed to identify mRNA expression of MYG1 and the key downstream transcription factors HES1， HES5， and HEY1 of the Notch signaling pathway， as well as the protein expression of NICD1. Co?transfection plasmids （for over?expressing STK4?AS1 and MYG1） were used to detect the mRNA expression of HES1， HES5， and HEY1 and the protein expression of NICD1 which acted as the key downstream transcription factors in the Notch signaling pathway， as well as the effects on the proliferation， migration， and invasion abilities of ESCC cells. Results The expression of STK4?AS1 was decreased in ESCC cell lines （P < 0.01）. Over?expression of STK4?AS1 inhibited the proliferation， migration and invasion abilities in Eca109 and Kyse150 cells （P < 0.05）. STK4?AS1 negatively regulated the expression of MYG1 （P < 0.01）， and the expression of MYG1 was increased in ESCC cell lines （P < 0.01）. Over?expression of MYG1 could partially reverse the effect of STK4?AS1 on the malignant biological behavior of Eca109 and Kyse150 cells （P < 0.05）， as well as the mRNA expressions of HES1， HES5， and HEY1 and the protein expression of NICD1 （P < 0.05）. Conclusion STK4?AS1 affects the malignant biological behaviors of ESCC through the MYG1/Notch signaling pathway。