Efficient interhemispheric integration of neural activity between left and right primary motor cortex (M1) is critical for inter-limb motor control. We employed optogenetic stimulation to establish a framework for probing transcallosal M1–M1 interactions in rats. We performed optogenetic stimulation of excitatory neurons in right M1 of male Sprague-Dawley rats. We recorded the transcallosal evoked potential in contralateral left M1 via chronically implanted electrodes. Recordings were performed under anesthesia combination of dexmedetomidine and a low concentration of isoflurane. We systematically varied the stimulation intensity and duration to characterize the relationship between stimulation parameters in right M1 and the characteristics of the evoked intracortical potentials in left M1. Optogenetic stimulation of right M1 consistently evoked a transcallosal response in left M1 with a consistent negative peak (N1) that sometimes was preceded by a smaller positive peak (P1). Higher stimulation intensity or longer stimulation duration gradually increased N1 amplitude and reduced N1 variability across trials. A combination of stimulation intensities of 5–10 mW with stimulus durations of 1–10 ms were generally sufficient to elicit a robust transcallosal response in most animal, with our optic fiber setup. Optogenetically stimulated excitatory neurons in M1 can reliably evoke a transcallosal response in anesthetized rats. Characterizing the relationship between “stimulation dose” and “response magnitude” (i.e., the gain function) of transcallosal M1-to-M1 excitatory connections can be used to optimize the variables of optogenetic stimulation and ensure stimulation efficacy.