Recently, we showed that increased SLPI levels prevent HPV-infections and metastasis in smoking-induced, non-HPV-driven HNSCC. Here we focus on the role of SLPI in non-HPV-driven HNSCC, investigating tumor tissue and non-neoplastic mucosa from the same patients and from non-HNSCC patients. Gene and protein expression of SLPI and gene expression of annexin 2 (a SLPI receptor), nicotine receptor (α7AChR) and arylhydrocarbon receptor (AhR) were analyzed in HNSCC patients (20 smokers; 16 non-smokers). SLPI-results were correlated with the patients' HPV-status. Non-neoplastic mucosa of HNSCC patients and normal mucosa from non-HNSCC individuals (18 smokers; 20 non-smokers) was analyzed for the same parameters. Tissue of the inferior turbinate (n=10) was incubated with nicotine for analysis of the same genes. SLPI gene expression in tumor tissue was 109.26±23.08 times higher in smokers versus non-smokers. Non-neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49±1.89-fold non-HNSCC; 18.02±3.93-fold HNSCC patients). Annexin 2 gene expression was also increased in smokers. SLPI data were corroborated by immunohistochemistry. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r2=0.15, p<0.001) was shown ex vivo. Nicotine and smoking increased α7AChR and AhR gene expression. Five patients, showing no/low SLPI expression, were HPV16-positive. A significant correlation between smoking and SLPI expression in tumors and to our knowledge for the first time in mucosa of HNSCC and non-HNSCC patients was established. Together with the finding that all patients with HPV-infection showed no/low SLPI expression, these data support our intriguing hypothesis that smoking induced up-regulated SLPI prevents HPV-infections. © 2013 Wiley Periodicals, Inc.