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Ankylosing spondylitis (AS) is mostly characterized by inflammation of the sacroiliac joints, enthesis and the spine. This study aims to determine gene expression profile of proinflammatory cytokines and their correlations with disease activity, spinal mobility, functional status, quality of life, and smoking in Iranian patients with ankylosing spondylitis. Peripheral blood mononuclear cells (PBMCs) were isolated from 48 patients with AS and 47 age and gender-matched healthy controls; then total RNA content of leukocytes was extracted, followed by cDNA synthesis from the mRNA of PBMCs. Quantitative polymerase chain reaction was performed to measure mRNA expression of TNF-α, IL-1β and IFN-γ genes. Clinical characteristics were evaluated and their correlations were analyzed with gene expression levels of the prionflammatory cytokines. A significant overexpression of TNF-α was observed in the patient group, but there was no significant difference in expression of other cytokines between groups. A positive correlation (P< 0.01) between TNF-α and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and negative correlation (P< 0.05) between IFN-γ and Bath Ankylosing Spondylitis Functional Index (BASFI) were found. Eta-Squared test supported significant results about the smoking effect on Bath Ankylosing Spondylitis Metrology Index (BASMI) (P< 0.05) and IFN-γ (P< 0.01). Our results demonstrated that TNF-α was the most important cytokine responsible for inflammatory-related outcomes of AS in the Iranian population.