Natural killer (NK) cells participate in the immune response against infection and cancer. An emerging body of epidemiological data supports that circadian homeostasis may constitute a factor risk for cancer development. Physiological rhythms under circadian control persist in the absence of light entrainment and ultimately rely on a molecular clock. We have previously shown that NK cell cytolytic activity follows a daily rhythm and that NK cells enriched from light-entrained rats present 24-h oscillations of clock genes, cytolytic factors, and cytokines. To investigate whether these oscillations are under a genuine circadian control, we assessed the daily expression of clock genes (Per1, Per2, Clock, and Bmal1), a clock-controlled gene (Dbp), cytolytic factors (granzyme B and perforin), and cytokines (IFN-gamma and TNF-alpha) in NK cells enriched from rats maintained in constant darkness (DD). In addition, we investigated whether the disruption of the NK cell clock by RNA interference (RNAi) affects the expression of cytolytic factors and cytokines. Persistent 24-h oscillations were found in the expression levels of clock genes, cytolytic factors, and cytokines in NK cells enriched from DD rats. In addition, RNAi-mediated Per2 knockdown caused a significant decrease of granzyme B and perforin levels in the rat derived NK cell line RNK16. Taken together, these results provide evidence supporting that NK cell function is under circadian regulation.