Isocorydine (ICD) exhibits strong antitumor effects on numerous human cell lines. However, the anticancer activity of ICD against oral squamous cell carcinoma (OSCC) has not been reported. The anticancer activity, migration and invasion ability, and changes in the cytoskeleton morphology and mechanical properties of ICD in OSCC were determined. Changes in the contents of reactive oxygen species (ROS), the mitochondrial membrane potential (MMP), ATP, and mitochondrial respiratory chain complex enzymes Ⅰ-Ⅳ in cancer cells were studied. ICD significantly inhibited the proliferation of oral tongue squamous cells (Cal-27), with an IC50 of 0.61 mM after 24 h of treatment. The invasion, migration, and adhesion of cancer cells were decreased, and cytoskeletal actin was deformed and depolymerized. In comparison to an untreated group, the activities of mitochondrial respiratory chain complex enzymes I-IV were significantly decreased by 50.72%, 27.39%, 77.27%, and 73.89%, respectively. The ROS production increased, the MMP decreased by 43.65%, and the ATP content decreased to 17.1 ± 0.001 (mmol/mL); ultimately, the apoptosis rate of cancer cells increased up to 10.57% after 24 h of action. These findings suggest that ICD exerted an obvious anticancer activity against OSCC and may inhibit Cal-27 proliferation and growth by causing mitochondrial dysfunction and interrupting cellular energy.