Biological networks consist of a defined set of regulatory motifs. Subcellular compartmentalization of regulatory molecules can provide a further dimension in implementing regulatory motifs. However, spatial regulatory motifs and their roles in biological networks have rarely been explored. Here we show, using experimentation and mathematical modeling, that spatial segregation of GIGANTEA (GI), a critical component of plant circadian systems, into nuclear and cytosolic compartments leads to differential functions as positive and negative regulators of the circadian core gene, LHY, forming an incoherent feedforward loop to regulate LHY. This regulatory motif formed by nucleocytoplasmic partitioning of GI confers, through the balanced operation of the nuclear and cytosolic GI, strong rhythmicity and robustness to external and internal noises to the circadian system. Our results show that spatial and functional segregation of a single molecule species into different cellular compartments provides a means for extending the regulatory capabilities of biological networks.