The DNA damage response is critical for maintaining genome integrity and preventing damage to DNA due to endogenous and exogenous insults. Mitomycin C (MMC), a potent DNA cross-linker, is used as a chemotherapeutic agent because it causes DNA inter-strand cross-links (DNA ICLs) in cancer cells. While many microRNAs, which may serve as oncogenes or tumor suppressors, are grossly dysregulated in human cancers, little is known about their roles in MMC-treated lung cancer. Here, we report that miR-128-3p can attenuate repair of DNA ICLs by targeting SPTAN1 (αII Sp), resulting in cell cycle arrest and promoting chromosomal aberrations in lung cancer cells treated with MMC. Using computational prediction and experimental validation, SPTAN1 was found to be a conserved target of miR-128-3p. We then found that miR-128-3p caused translational inhibition of SPTAN1, reducing its protein level. SPTAN1 repression via miR-128-3p also induced cell cycle arrest and chromosomal instability. Additionally, miR-128-3p significantly influenced interaction of the αII Sp/FANCA/XPF complex, thus limiting DNA repair. In summary, the results demonstrate that miR-128-3p accelerates cell cycle arrest and chromosomal instability in MMC-treated lung cancer cells by suppressing SPTAN1, and these findings could be applied for adjuvant chemotherapy of lung cancer.