Although circadian oscillation in dynamics of intracellular Ca2+ signals has been observed in both plant and animal cells, it has remained unknown whether Ca2+ signals play an in vivo role in cellular oscillation itself. To address this question, we modified the dynamics of intracellular Ca2+ signals in circadian pacemaker neurons in vivo by targeted expression of varying doses of a Ca2+ buffer protein in transgenic Drosophila melanogaster. Intracellular Ca2+ buffering in pacemaker neurons results in dose-dependent slowing of free-running behavioral rhythms, with average period >3 h longer than control at the highest dose. The rhythmic nuclear accumulation of a transcription factor known to be essential for cellular circadian oscillation is also slowed. We also determined that Ca2+ buffering interacts synergistically with genetic manipulations that interfere with either calmodulin or calmodulin-dependent protein kinase II function. These results suggest a role for intracellular Ca2+ signaling in regulating intrinsic cellular oscillation in vivo.