The upregulation of tribbles homolog 3 (TRB3), a pseudokinase in mammals, has been observed in several types of malignant cancer, including thyroid, ovarian, liver and colorectal cancer. However, the pathological role and the regulatory mechanism of TRB3 in cancer remain unknown. In the current study, we demonstrated that the expression of TRB3 was upregulated in non-small cell lung cancer (NSCLC), correlating with tumor metastasis, disease recurrence and poor survival in patients. Knocking down TRB3 in aggressive lung cancer cell lines was demonstrated to significantly inhibit their malignant behaviors, including in vitro invasion and cell proliferation, as well as in vivo metastasis and tumor growth. The correlation between TRB3 and Notch 1 expression revealed that Notch 1 was downregulated by the knockdown of TRB3 in the lung adenocarcinoma cell lines. These results have provided insights into the correlation between TRB3 expression and lung cancer progression, and thus may have potential for the prognosis and therapy of lung cancer.