Matrix-metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) expression levels have been demonstrated to have prognostic value in oral squamous cell carcinoma (OSCC). The present study hypothesized that melatonin, a small lipophilic molecule primarily secreted by the pineal gland, may be able to regulate MMP activity in OSCC progression. This study aimed to investigate the associations between melatonin, MMPs, TIMPs and the histopathological characteristics of patients with OSCC. A total of 40 men with OSCC (mean age, 57±7 years) and 30 healthy men (mean age, 56±5 years) were enrolled in the present study. Enzyme immunoassays were used to measure the serum levels of melatonin, MMP-9, MMP-2, TIMP-1 and TIMP-2 before and after transoral surgery for OSCC. Serum melatonin level was significantly lower in patients with OSCC compared with controls, both pre-surgery and 2 days after surgery (P<0.001). In addition, melatonin level was negatively correlated with MMP-9 (r=-0.6371) and the MMP-9/TIMP-1 ratio (r=-0.4700), but not with the MMP-2 or MMP-2/TIMP-2 ratio, in patients with OSCC. These results demonstrated that low levels of melatonin and high levels of MMP-9 correlated with large tumors with invasive depth (r=-0.35 and r=0.33) and lymph node metastasis (r=-0.56 and r=0.34). The results of this retrospective clinical study suggested that melatonin may be considered as a predictive biomarker of tumor growth and metastasis and a potential therapeutic agent for patients with OSCC.