Objective(s)Tracheostomy‐associated granulation tissue is a common, recurrent problem occurring secondary to chronic mucosal irritation. Although granulation tissue is composed of predominantly innate immune cells, the phenotype of monocytes and macrophages in tracheostomy‐associated granulation tissue is unknown. This study aims to define the myeloid cell population in granulation tissue secondary to tracheostomy.MethodsGranulation tissue biopsies were obtained from 8 patients with tracheostomy secondary to laryngotracheal stenosis. Cell type analysis was performed by flow cytometry and gene expression was measured by quantitative real‐time polymerase chain reaction. These methods and immunohistochemistry were used to define the monocyte/macrophage population in granulation tissue and were compared to tracheal autopsy control specimens.ResultsFlow cytometry demonstrated macrophages (CD45+CD11b+) and monocytes (CD45+FSClowSSClow) represent 23.2 ± 6% of the granulation tissue cell population. The M2 phenotype (CD206) is present in 77 ± 11% of the macrophage population and increased compared to the M1 phenotype (p = 0.012). Classical monocytes (CD45+CD14highCD16low) were increased in granulation tissue compared to controls (61.2 ± 7% and 30 ± 8.5%, p = 0.038). Eighty‐five percent of macrophages expressed pro‐inflammatory S100A8/A9 and 36 ± 4% of macrophages co‐localized CD169, associated with tissue‐resident macrophages. M2 gene expression (Arg1/CD206) was increased in granulation tissue (3.7 ± 0.4, p = 0.035 and 3.5 ± 0.5, p = 0.047) whereas M1 gene expression (CD80/CD86) was similar to controls (p = 0.64, p = 0.3). Immunohistochemistry of granulation tissue demonstrated increased cells co‐localizing CD11b and CD206.ConclusionsM2 macrophages are the dominant macrophage phenotype in tracheostomy‐associated granulation tissue. The role of this cell type in promoting ongoing inflammation warrants future investigation to identify potential treatments for granulation tissue secondary to tracheostomy.Level of Evidence3 Laryngoscope, 133:2346–2356, 2023