Abstract Cervical cancer is the most frequent cause of gynecologic cancer–associated death worldwide. Animal models that demonstrate metastatic patterns consistent with the clinical course of cervical cancer are urgently needed to conduct studies focused on understanding the mechanisms of the disease and identifying optimal treatments. To address this, we established an orthotopic xenograft model of cervical cancer in female NOD-SCID mice using SiHa and ME180 cell lines stably expressing green fluorescent protein to evaluate the role of microRNA-21 (miR-21) in spontaneous lymph node metastasis in vivo. In this case, SiHa and ME180 cells were transduced by lentivirus to stably express green fluorescent protein and miR-21. Overexpression of miR-21 promoted proliferation, migration, and invasion of SiHa and ME180 cells in vitro. Finally, an orthotopic xenograft model of human cervical cancer was successfully established in NOD-SCID mice. Using this model, we confirmed that overexpression of miR-21 resulted in an increase in the size of primary tumors and in the frequency of spontaneous lymph node metastasis at the time of excision. Therefore, the use of the orthotopic xenograft model should allow for the investigation of novel factors that affect metastasis of cervical cancer and presents an opportunity to evaluate potential therapeutic agents that may inhibit the spread of the disease.