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Background: Examinations on blood samples and gingival crevicular fluid of subjects with chronic periodontitis showed that smoking increased production of cytokines. Objective: To evaluate the expression of immune markers on mononuclear inflammatory cell infiltrate at periodontitis and healthy tissue among smoking and non-smoking subjects. Methods: This case-control study was performed on 41 patients who referred to a clinic of periodontology at a Mashhad dental school in Iran in 2016. The participants were all of Iranian Khorasanian ethnicity with age range of 35-65 years. Gingival biopsies were obtained during routine periodontal flap procedure. Immunohistochemistry using markers of CD20, CD3, CD68, and CD45RO was carried out. Data was analyzed by SPSS version 15, using one-way ANOVA and Tukey HSD test. Results: Nonsmoker subjects showed significantly greater numbers of CD20+, CD68+, CD3+ cells compared to smoker subjects, both at healthy and periodontitis tissue biopsies (p<0.00), whereas there was no significant difference in terms of CD45RO (p=0.120). Conclusion: Cigarette smoking, results in infiltrative mononuclear chronic inflammatory cells reduction in connective periodontium