Circadian rhythms control many aspects of our daily lives: from waking and sleeping, to cell cycling within the body’s tissues. The suprachiasmatic nucleus (SCN) is often referred to as the body’s master pacemaker due to its ability to receive light information from the optic nerve and modulate a transcriptional-translational feedback loop, known as the molecular clock, and project circadian information from the SCN to the rest of the body via neuropeptide regulation. VAX1 and SIX3 are two homeodomain transcription factors, known to regulate gene expression, that are highly expressed in the mature mouse SCN. Through the use of transgenic mouse models, we developed conditional Vax1 and Six3 knockout mice bred with Per2-luciferase reporters, a molecular clock gene, to monitor the effects of VAX1 and SIX3 deletion on SCN circadian output. We find that deletion of Vax1 or Six3 from the adult mouse SCN results in shorter and longer circadian period, respectively. Additionally, we determine the ability of VAX1 and SIX3 to increase the expression of Per2-luciferase in vitro, and additionally the ability of VAX1 to drive Vip-luciferase expression, the primary SCN neuropeptide responsible for projecting circadian information from the SCN to the body. Homeodomain transcription factors are known to regulate gene expression by directly binding to ATTA sites within the gene’s promoter. We identified ATTA sites within regulatory regions of Per2 and Vip genes as potential targets for direct VAX1 interaction and regulation of gene expression. Overall, these data collectively identify VAX1 and SIX3 as novel proteins that are responsible for maintaining proper circadian rhythms during adulthood.