Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.