Cyclin-dependent kinase (CDK) inhibitor p27Kip1 is a diagnostic and prognostic marker of various malignancies. Low expression of p27Kip1 reflects poor prognosis, and an inverse correlation between the expression of p27Kip1 and degree of tumor malignancy has been reported. Because p27Kip1 mutation is extremely rare in human tumors, expression of p27Kip1 protein is thought to be controlled by post-transcriptional mechanism involved in inactivator, S-phase kinase associated protein 2 (Skp2) and Jun activation domain-binding protein 1 (Jab1). In this study, we investigated the role of gene therapy targeting p27Kip1 in human head and neck cancer cells. Human head and neck cancer (HNt and HSY) cells expressed p27Kip1, Skp2 and Jab1. To determine the function of p27Kip1, Skp2 and Jab1, we transfected head and neck cancer cells with pcDNA3.1-p27Kip1 wt, pcDNA3.1-p27Kip1 mt or treated with Skp2 antisense oligonucleotide (AS) or Jab1 AS. The transfections or treatments inhibited the growth of HNt and HSY cells. The growth inhibition mediated by pcDNA3.1-p27Kip1 mt or Skp2 AS or Jab1 AS specifically due to a significant induction of apoptosis characterized by an increase in fragmentation of nuclei and activation of caspase-3. The transfection of pcDNA3.1-p27Kip1 mt or treatment with Skp2 AS and Jab1 AS induced a strong growth inhibition of xenograft tumors. These findings suggest that p27Kip1 mt, Skp2 AS and Jab1 AS have the potential to become a novel and powerful gene therapy tool, and that stability of p27Kip1 protein may improve therapeutic benefits to patients with head and neck cancer.