Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been found to be involved in multiple cancers. However, the effect of small interfering RNA (siRNA)‑induced knockdown of TRAF6 on the biological behaviors of cancer cells remains unknown. Thus, the present study aimed to investigate the effect of siRNA-induced knockdown of TRAF6 on the biological behaviors of human lung cancer SPC-A1 cells. The expression of TRAF6 was determined in human lung adenocarcinoma A549, non-small cell lung cancer H1650, human airway epithelial Calu-3 and human lung cancer SPC-A1 cell lines using quantitative RT-PCR (qRT‑PCR) and western blotting at the transcriptional and translational levels. TRAF6 expression was knocked down in the SPC-A1 cells using an siRNA technique, and the effects of TRAF6 knockdown on NF-κB activity, cell proliferation, apoptosis, cell cycle, invasion and migration of the SPC-A1 cells were determined using electrophoretic mobility shift assay (EMSA), cell proliferation assay, flow cytometry, Transwell invasion assay and scratch wound assay. In addition, the protein expression of CD24, CXCR4, MMP1, MMP2, MMP9, TWIST, TIMP-2 and Slug was quantified using western blotting assay. Western blotting and qRT-PCR assays showed upregulation of TRAF6 at both the translational and transcriptional levels in the Calu-3 and SPC-A1 cells, and K63-linked ubiquitination of TRAF6 and constitutive NF-κB activation were detected in the SPC-A1 cells. Knockdown of TRAF6 inhibited the migration and invasion and promoted the apoptosis of the SPC-A1 cells, but had little effect on cell proliferation and the cell cycle. In addition, siRNA-induced TRAF6 knockdown caused a marked reduction in the protein expression of CD24 and CXCR4, but had little effect on MMP-1, MMP-2, MMP-9, Twist, TIMP-2 or Slug expression. The present study demonstrated that TRAF6 is upregulated in human lung cancer cells, and siRNA-induced TRAF6 knockdown inhibits the invasion of lung cancer cells and promotes apoptosis. It is suggested that TRAF6 may be a promising target for the therapy of lung cancer.