Circadian systems regulate the immune system by various molecular and physiological pathways. Disruption to the circadian temporality of these pathways is associated with disease formation and progression. Circadian clock genes have been shown to regulate pathways involved in cellular proliferation, apoptosis, and DNA damage response, as aberrant rhythms in these genes are associated with various diseases. However, there is growing evidence that specific circadian genes differentially regulate functional pathways of immunocompetent cells. To extend our previous findings of the role of Period 2 in regulating splenocyte rhythms, we report that mice carrying a mutation in the Period 1 gene (Per1^−/− mice), involved in the negative limb of the molecular clock, display significantly altered rhythms of cytokine (eg, interferon-γ) and cytolytic factors (eg, perforin and granzyme B) in splenic natural killer (NK) cells. Altered rhythms of NK cell immune factors were accompanied by changes in circadian expression of circadian clock genes, Bmal1 and Per2. In addition, Per1^−/− circadian running-wheel activity rhythms remained rhythmic during constant darkness, although with a shortened free-running circadian period, suggesting primary involvement of peripheral molecular clocks. These findings indicate that the Per1 gene through NK cellular clocks modulates immune pathways.