Transcriptomics, proteomics, phosphoproteomics, and comprehensive multi-omics analysis were conducted to investigate the relationship between Plexin A1 (PLXNA1) and esophageal cancer (EC) cells. The positive rate of PLXNA1 in EC tissues was significantly high than in adjacent normal tissue. Knocking down PLXNA1 in EC cells led to increased apoptosis and decreased proliferation, invasion, and migration. Analyses revealed that PLXNA1 knockdown influenced cellular transcriptional regulation and cancer-related pathways. Elevated expression of PLXNA1 in patients with EC correlates with survival outcomes. PLXNA1 plays a crucial role in transcriptional regulation and cancer pathways in EC. The expression of PLXNA1 was elevated in patients with EC and might play a key role in EC by regulating the transcriptional process and carcinogenic pathways.