The circadian clock, located in the suprachiasmatic nucleus (SCN), receives a major afferent from the median raphe nucleus (MRN). In the Syrian hamster, only about 50% of the cells giving rise to this afferent contain serotonin. There is mixed evidence as to whether the serotonergic portion of this projection is involved in non-photic phase shifting of circadian locomotor rhythms. In order to better characterize the non-serotonergic projections, we conducted retrograde tract tracing using the beta subunit of cholera toxin combined with multi-label immunohistochemistry. Similar to previous findings, almost half of the retrogradely labeled cells contained serotonin. Additionally, approximately 30% of the retrogradely labeled cells contained vesicular glutamate transporter 3 (VGLUT3), but not serotonin. Surprisingly, some dorsal raphe cholera toxin labeling was also noted, particularly in animals with central-SCN injections. To determine if the non-serotonergic projections were important for non-photic phase shifts elicited by MRN stimulation, the MRN was electrically stimulated in animals pretreated with SCN injection of either the serotonin neurotoxin 5,7-dihydroxytryptamine or vehicle control. Intact animals phase advanced to midday electrical stimulation of the raphe while lesioned animals did not. Together, these results show that although some of the non-serotonergic raphe projections to the SCN contain VGLUT3, it is the serotonergic raphe innervation of the SCN that is critical for non-photic phase shifting elicited by MRN stimulation.