The novel (nua) kinase family 1 (NUAK1) is an AMPK-related kinase and its expression is associated with tumor malignancy and poor prognosis in several types of cancer, suggesting its potential as a target for cancer therapy. Therefore, the development of NUAK1-targeting inhibitors could improve therapeutic outcomes in cancer. We synthesized KI-301670, a novel NUAK1 inhibitor, and assessed its anticancer effects and mechanism of action in pancreatic cancer. It effectively inhibited pancreatic cancer growth and proliferation, and induced cell cycle arrest, markedly G0/G1 arrest, by increasing the expression of p27 and decreasing expression of p-Rb and E2F1. Additionally, the apoptotic effect of KI-301670 was observed by an increase in cleaved PARP, TUNEL-positive cells, and annexin V cell population, as well as the release of cytochrome c via the loss of mitochondrial membrane potential. KI-301670 inhibited the migration and invasion of pancreatic cancer cells. Mechanistically, KI-301670 effectively inhibited the PI3K/AKT pathway in pancreatic cancer cells. Furthermore, it significantly attenuated tumor growth in a mouse xenograft tumor model. Our results demonstrate that a novel NUAK1 inhibitor, KI-301670, exerts anti-tumor effects by directly suppressing cancer cell growth by affecting the PI3K/ AKT pathway, suggesting that it could be a novel therapeutic candidate for pancreatic cancer treatment.