Background: The diagnosis of microinvasive laryngeal squamous cell carcinoma (LSCC) is not always straightforward and sometimes can be very challenge in daily clinical practice. The focus lies in the confirmation of microinvasion. Cancer-associated fibroblasts (CAFs), as the major element of reactive tumor stroma, are believed to participate actively in the growth and invasion of tumor cells. Objectives: To evaluate the diagnostic role of alpha-smooth muscle actin (alpha-SMA) labelling CAFs in microinvasive LSCC. Methods: A total of 81 laryngeal biopsy specimens were retrieved, including 41 cases of microinvasive LSCC with depth of invasion no more than 3 mm, 20 laryngeal squamous intraepithelial lesion (SIL), and 20 benign pseudoepitheliomatous hyperplasia (PEH). All cases were stained for immunohistochemistry, using antibody against the alpha-SMA antigen. The correlation between the presence of CAFs in microinvasive LSCC and tumor histological characteristics was investigated. Results: Immunoreactivity of alpha-SMA was detected in twenty-nine microinvasive LSCC (29/41, 71%), while no reactivity was observed in laryngeal SIL (0/20, 0%), and rarely in PEH (2/20, 10%). The alpha-SMA expression pattern in stroma of microinvasive LSCC was significantly different from that of SIL (chi(2(OBJ)) = 26.966, p = 0.000) and PEH (chi(2(OBJ)) = 19.838, p = 0.000). In addition, there seemed to be a certain correlation between the histological characteristics of microinvasive LSCC and the presence of interstitial CAFs. Conclusions: This study highlights the practical role of utilizing alpha-SMA in the pathological diagnosis of micro invasive LSCC, with emphasis on variable histomorphologic features of microinvasive LSCC.