Rhomboid domain containing 1 (RHBDD1) gene, which was reported to be upregulated in human several cancer, was associated with carcinogenesis. However, the potential biological function of RHBDD1 in non-small cell lung cancer (NSCLC) carcinogenesis remains still not known. In this study, we aimed to investigate the role of RHBDD1 and its underlying molecular mechanism in NSCLC. The gene RHBDD1 expression was detected in NSCLC tissues and matched nontumor adjacent tissues. In vitro experiments, NSCLC cell lines (A549, H1650, H358 and H1299) were performed to investigate the biological function of RHBDD1 and its molecular mechanism. Our findings showed that the mRNA and protein expression levels of RHBDD1 were notably increased in human NSCLC tissues and cell lines, especially in A549 and H1650 cells. Moreover, silencing of RHBDD1 by RNAi notably inhibited NSCLC cell proliferation and increased cell apoptosis. Caspase-3/7 activity was remarkably increased in cells treated with RHBDD1 siRNA. RHBDD1 silencing notably reduced the number of invading cells. Furthermore, our findings showed that silencing of RHBDD1 notably inhibited the mRNA and protein expression levels of ZEB1 in A549 and H1650 cells. The phosphorylation of PI3K and AKT was also remarkably decreased by RHBDD1 silencing. ZEB1/AKT overexpression reversed the effect of RHBDD1 silencing on NSCLC cell growth and invasion. Taken together, our findings indicated that RHBDD1 silencing inhibited cell growth and invasion of non-small cell lung cancer by mediating ZEB1/PI3K/AKT signaling pathway, implying that RHBDD1 was possibly a potential diagnostic and therapeutic target for NSCLC treatment.