Background: Macrophages are important cells for the innate immunity. Circulating monocytes are attracted to tissues by chemotactic factors and become macrophages under the influence of their microenvironment. Several studies suggested that local and systemic upregulation of fibrogenic cytokines and downregulation of antifibrotic cytokine are central to the pathogenesis of oral submucous fibrosis (OSMF). Currently, there have been no attempts made to elucidate the presence and role of macrophages in OSMF. Aim: Our aim was to study the expression of CD68 in OSMF patients and to investigate the possible correlation of macrophages using CD68 in various histopathological grades of OSMF. Subjects and Methods: A prospective case–control study which included 40 patients was conducted after obtaining informed consent and Ethical Committee clearance. Ten cases were normal control and thirty cases had OSMF. Biopsy was performed and a quantitative study of macrophages was done using CD68 antigen and was immunohistochemically localized. Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) 17.0 version (SPSS Inc., Chicago, IL, USA). Results: OSMF was observed in male patients of a younger age group. The macrophage number in the patients of intermediate and advanced stage of OSMF was higher than that of the controls which was statistically significant (P < 0.05). Conclusion: The findings of this study suggest that CD68 plays a vital role in the pathogenesis of OSMF and can be regarded as a useful marker for assessing the progress of the disease.