PURPOSE: The flexible protein GRB2 interacts with HER1-4 on the cell surface and regulates the development of tumor cells; meanwhile, it is also an RBP that plays an important role in post-transcriptional regulation in eukaryotes, which affects every stage of mRNA synthesis, modification, splicing, and stabilization. Although some studies have found a connection between GRB2 and HER2-overexpression breast cancer, highlighting the potential of GRB2 as a novel biomarker that stimulates tumor growth, limited data were available to elaborate on their interaction mechanisms. METHODS: In this research, we found 396 different gene expressions between the Grb2-knockdown group and the SK-BR-3 group by the RNA sequencing approach. After GRB2 was knocked down, 956 alternative splicing events occurred. RESULTS: The fRIP-seq results showed that GRB2-binding reads were significantly enriched in the intron region, indicating that UUAGC and UUGGUUGG might be the binding motifs. An integration analysis of DEGs with the peak genes of fRIP-seq revealed that 63 genes possess GRB2 binding sites on their mRNAs or antisense RNAs. By integration analysis of AS events with the peak genes of fRIP-seq, 66 genes related to AS events were found. CONCLUSIONS: Above, these AS events may be regulated by GRB2 to promote the progression of HER2-overexpression breast cancer.