Non-small cell lung cancer (NSCLC) patients carrying HER2 20ins exon 20 insertion (HER2 20ins) are poorly respond to conventional therapies and pan HER inhibitors. Trastuzumab Deruxtecan (T-DXd) has been approved in second line treatment in advanced NSCLC, but its efficacy in operable patients remains unclear. Here, we report a case of a patient with operable, HER2 20ins NSCLC who received neoadjuvant T-DXd. We utilized whole-genome sequencing (WGS) and immune microenvironment analysis to investigate the treatment's impact. WGS analysis revealed significant reduction in tumor clones with genome instability and extrachromosomal DNA (ecDNA) following T-DXd treatment. Meanwhile, immune profiling demonstrated substantially increased CD8 + T-cell infiltration in tumor cores with elevated PD1 expression. In contrast, conventional chemotherapy did not significantly alter CD8 + T cell infiltration or PD1 expression in another matched HER2 20ins patient. These findings suggest that neoadjuvant T-DXd may offer a well-tolerated and efficient therapeutic strategy for locally advanced NSCLC with HER2 20ins mutations.