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Abstract Background Lung cancer (LC) is one of the leading causes of death worldwide. Programmed cell death receptor 1 (PD-1) interacts with its ligand (PDL-1) on T cells inhibiting its functioning which may affect the patient's immunological response. Aim Investigate if there is a link between smoking and tissue expression of PDL-1 and vitamin D receptor (VDR) in lung cancer patients. In addition, the relation of vitamin D with smoking and these biochemical markers. Methods PDL-1 and VDR expressions were evaluated by real-time PCR in 54 lung cancer biopsy samples and 36 controls to prove this hypothesis. Vitamin D levels in the blood were measured using an ELISA. Results Expressions of PDL-1 were significantly upregulated in LC patients than in controls. The highest expression was in stage II and in squamous cell carcinoma followed by small cell carcinoma then adenocarcinoma. However, VDR expressions and vitamin D levels in serum were significantly downregulating in LC patients than in controls. There was a positive correlation between PDL-1expression and duration of smoking but not smoking index. Also, there is an inverse correlation between duration of smoking, smoking index, and VDR. Conclusion Expression of PDL-1 in LC was significantly upregulated and correlated with staging. Interestingly, our current study for the first time explained the role of duration of smoking on PDL-1 and VDR in the pathogenesis of LC. As PDL-1 expression increased with duration of smoking whereas VDR decreased, this novel findings may provide a possible link between the cumulative effect of smoking and the level of expressions of these biomarkers.