Temporal Interference Stimulation (TIS) represents a novel non-invasive brain stimulation technique that deeply targets specific brain regions using the differential beat frequency of two high-frequency stimulation pairs. This study investigated the neuromodulatory effects of TIS at different beat frequencies on cortical excitability in the rat motor cortex. Rats were randomly assigned into four groups, receiving TIS at alpha (10 Hz), beta (20 Hz), gamma (70 Hz), or sham frequencies targeting the motor cortex for 20 min under anesthesia. Cortical excitability and inhibition were evaluated by measuring motor-evoked potentials (MEPs), input-output (I/O) curves, and long-interval intracortical inhibition (LICI) before and after TIS. Additionally, immunohistochemistry was performed for neural biomarkers c-Fos and glutamic acid decarboxylase (GAD-65) to confirm targeted neural activation following TIS. We also examined glial fibrillary acidic protein (GFAP)-positive cells in the stimulated region to assess astrocyte responses associated with TIS. Alpha and gamma TIS significantly increased MEP amplitudes compared to sham stimulation. The analysis of I/O curves revealed a significant enhancement in the area under the curve (AUC) post-stimulation in the alpha and gamma TIS groups. Notably, only gamma TIS significantly reduced intracortical inhibition, indicated by an increased LICI ratio post-stimulation. Immunohistochemical analysis demonstrated a significant 35% increase in c-Fos-positive cells in the stimulated motor cortex regions after TIS compared to sham, whereas no significant changes in GAD-65-positive cells or GFAP expression were observed. These findings indicate that a single session of alpha or gamma TIS effectively modulates cortical excitability, highlighting its potential for targeted neuromodulation applications.