In the present paper, we have further developed an in vitro model to study neuronal-glial interaction at trigeminal level by characterizing the effects of conditioned medium (CM) collected from activated primary cultures of satellite glial cells (SGCs) on calcitonin gene-related peptide (CGRP) release from rat trigeminal neurons. Moreover, we investigated whether such release is inhibited by a clinically relevant anti-migraine drug, sumatriptan. CM effects were tested on trigeminal neuronal cultures in different conditions of activation and at different time points. Long-term exposures of trigeminal neurons to CM increased directly neuronal CGRP release, which was further enhanced by the exposure to capsaicin. In this framework, the anti-migraine drug sumatriptan was able to inhibit the evoked CGRP release from naïve trigeminal neuron cultures, as well as from trigeminal cultures pre-exposed for 30 min to CM. On the contrary, sumatriptan failed to inhibit evoked CGRP release from trigeminal neurons after prolonged (4 and 8 h) pre-exposures to CM. These findings were confirmed in co-culture experiments (neurons and SGCs), where activation of SGCs or a bradykinin priming were used. Our data demonstrate that SGCs activation could influence neuronal excitability, and that this event affects the neuronal responses to triptans.