Cordycepin is the major functional component of Cordyceps species and is widely used in traditional oriental medicine. Cordycepin has been shown to possess many pharmacological properties, such as enhancement of immune function along with anti-inflammatory, antioxidant, anti-aging, and anti-cancer effects. Here, we investigated the inhibitory effects of cordycepin on cell migration and invasion, which are two critical cellular processes that are often deregulated during metastasis, using HCT116 human colorectal carcinoma cells. According to our data, cordycepin at non-cytotoxic concentrations markedly inhibited the motility and invasiveness of HCT116 cells in a time-dependent manner. RT-PCR and Western blotting results indicated that cordycepin reduced the levels of claudin proteins, which are major components of tight junctions (TJs), and induced tightening of TJs. Cordycepin also attenuated the expression and activities of matrix metalloproteinases (MMPs)-2 and -9, whereas levels of tissue inhibitor of metalloproteinases (TIMPs)-1 and -2 were simultaneously elevated. These findings suggest that cordycepin reduces the migration and invasion of HCT116 cells by modulating the activities of TJs and MMPs.