Many studies have shown that the hyperactivation of ribosome biogenesis plays essential roles in the initiation and progression of cancers. As a ribosome assembly factor, PNO1 plays an important role in ribosome biogenesis. However, little is known about the expression and function of PNO1 in human tumors. In our present study, we aimed to explore the functional roles and the underlying molecular mechanisms of PNO1 in human lung adenocarcinoma (LUAD). Both bioinformatics databases and tumor tissues demonstrated that the expression of PNO1 in LUAD tissues was higher than that in adjacent tissues and predicted poor survival in LUAD patients. In vitro and in vivo assays suggested that downregulation of PNO1 expression suppressed LUAD cell proliferation and invasion. Further studies found that miR-340-5p depressed PNO1 expression via direct binding to the 3' untranslated region (UTR) of PNO1. PNO1 expression was negatively correlated with miR-340-5p expression in LUAD cells and tissue samples. Moreover, upregulation or downregulation of miR-340-5p expression reversed the effects of PNO1 inhibition and overexpression, respectively. Meanwhile, downregulation of PNO1 inhibited Notch signaling pathway which modulated epithelial mesenchymal transition (EMT). These results indicate that PNO1, negatively regulated by miR-340-5p, played an important role in LUAD progression via Notch signaling pathway. The miR-340-5p/PNO1/Notch axis might be a potential target for individualized and precise treatment of LUAD patients in the future.