Trans-(±)-kusunokinin (KU), a potential anticancer agent, has been reported as an AKR1B1 inhibitor, leading to the inhibition of oxidative stress and alteration of EMT-related proteins in aggressive breast cancer. Overexpression of AKR1B1 is associated with a poor prognosis in non-small cell lung cancer (NSCLC). Therefore, we aimed to elucidate the mechanism of action by which KU mediates its effects through AKR1B1 in NSCLC. The results indicate that KU inhibits the proliferation in a dose-dependent manner in both human A549 cells and cell lines derived from the AAV-CRISPR-Cas9 induced KrasP53 (KP) and KPLkb1 (KPL) mouse cancer models. Furthermore, KU inhibited cell migration in a dose-dependent manner under standard conditions as well as under high glucose treatment. Remarkably, KU suppressed AKR1B1 and SORD protein levels, reduced intracellular sorbitol and fructose and induced alterations in EMT-related proteins, such as ZEB1, E-cadherin, and vimentin, at a lower concentration than epalrestat (EP, AKR1B1 inhibitor). In an in vivo study, KU significantly prolonged the survival of mice carrying KPL lung tumors compared to the control group. Collectively, these findings suggest that KU inhibited the aggressive phenotype of lung cancers, highlighting the need for its clinical testing in NSCLC patients.