In this study, we described a transcriptome-wide m5C profile in ESCC for the first time, which showed aberrantly increased levels of RNA m5C modification in ESCC tumors due to the overexpression of NSUN2. NSUN2 plays a critical role in ESCC by positively regulating GRB2 through the m5C-LIN28B-based posttranscriptional regulation, while its own transcription is positively regulated by E2F1. Here, E2F1 is linked to esophageal squamous cell carcinoma.