Background <p>Aberrant alternative splicing (AS) generates multiple isoforms from a single gene, leading to functional diversity and potentially contributing to tumorigenesis. The spliceosome pathway is significantly activated in esophageal squamous cell carcinoma (ESCC). However, the functional implications and drug-response relevance of protein-coding and long non-coding RNA (lncRNA) AS and splicing factors (SFs) have not been fully characterized in ESCC.</p> Methods <p>The transcriptomic data (PRJNA533799, PRJNA435587, PRJNA704753, PRJNA489250, PRJNA298963) from Gene Expression Omnibus (GEO) database were used to summarize the AS landscape by rMATS analysis. Weighted correlation network analysis (WGCNA) and motif enrichment analysis were performed to investigate biological processes of AS events and SFs. The relationship between AS and drug sensitivity was explored by Spearman correlation analysis.</p> Results <p>A total of 3699 differential alternative splicing events (DASEs) were identified, predominantly involving protein-coding RNA and lncRNA (96.65%). The DASEs and SFs were primarily involved in biological processes such as the cell cycle, immune response, metabolism, metastasis, and apoptosis. Moreover, 85 RNA binding proteins were identified as dysregulated and hub SFs to modulate AS in ESCC. Notably, Bortezomib and BI-2536 were sensitive in inhibiting ESCC cell proliferation, correlating strongly with the DASEs and SFs.</p> Conclusion <p>Our research globally summarized the dysregulation of AS and SF, their associated biological effects, and recommended combined drug treatment strategies for ESCC. This study improves our knowledge of the molecular features of ESCC, supports the development of innovative therapies, and further advances fundamental research on precision medicine for ESCC.</p>