Oscillation regulates a wide variety of processes ranging from chemotaxis in Dictyostelium through segmentation in vertebrate development to circadian rhythms. Most studies on the molecular mechanisms underlying oscillation have focused on processes requiring a rhythmic change in gene expression, which usually exhibit a periodicity of >10 min. Mechanisms that control oscillation with shorter periods (<10 min), presumably independent of gene expression changes, are poorly understood. Oscillatory pollen tube tip growth provides an excellent model to investigate such mechanisms. It is well established that ROP1, a Rho-like GTPase from plants, plays an essential role in polarized tip growth in pollen tubes. In this article, we demonstrate that tip-localized ROP1 GTPase activity oscillates in the same frequency with growth oscillation, and leads growth both spatially and temporally. Tip growth requires the coordinate action of two ROP1 downstream pathways that promote the accumulation of tip-localized Ca2+ and actin microfilaments (F-actin), respectively. We show that the ROP1 activity oscillates in a similar phase with the apical F-actin but apparently ahead of tip-localized Ca2+. Furthermore, our observations support the hypothesis that the oscillation of tip-localized ROP activity and ROP-dependent tip growth in pollen tubes is modulated by the two temporally coordinated downstream pathways, an early F-actin assembly pathway and a delayed Ca2+ gradient-forming pathway. To our knowledge, our report is the first to demonstrate the oscillation of Rho GTPase signaling, which may be a common mechanism underlying the oscillation of actin-dependent processes such as polar growth, cell movement, and chemotaxis.