The aim of this study was to further clarify the functional mechanism of the triangular 67 (TRIM67) gene in lung cancer cells. We detected the expression of TRIM67 in lung cancer cells by RT-qPCR and Western blot, transfected si-NC, si-TRIM67, and pcDNA-ENAH into the cells. The expression of TRIM67 and ENAH was detected by Western blot and immunofluorescence localization, and CO-IP and GST pull-down experiments verified the interaction. Flow cytometry, Western blot, and transmission electron microscopy (TEM) evaluated the apoptosis and autophagy levels. TRIM67 was highly expressed in lung cancer cell lines. Knockdown of TRIM67 promoted apoptosis and autophagy of A549 and NCI-H1299 cells. TRIM67 interacted with the ENAH protein. ENAH restored the effect of knocking down TRIM67 and further inhibited apoptosis and autophagy of A549 and NCI-H1299 cells. TRIM67 inhibits apoptosis and autophagy of lung cancer cells by interacting with ENAH.