ABSTRACTp150glued is the largest subunit of dynactin protein complex, through which cargo vesicles link to the microtubule minus‐end directed motor protein dynein. In addition, p150glued also locates in the mother centriole where it organizes the subdistal appendage. The components of appendage are dynamically regulated throughout the cell cycle stages, but it is still unclear whether the centrosomal residency of p150glued correlated with cell cycle progression. Here we found that p150glued was located in the mother centriole during G1/S stage and its centrosomal residency was independent of microtubule transportation. However, the centrosomal p150glued became blurred at G2/M phase and this event was not regulated by its phosphorylation. Entering into mitosis, p150glued was robustly enriched in the mitotic spindle nearby the spindle poles but not in the centrosome. During serum starvation (G0 stage), p150glued appeared at the base of primary cilium and its depletion attenuated starvation‐induced primary cilium formation. We also checked its role in the maintenance of centrosome homeostasis and configuration, and found depletion of p150glued did not induce centrosome amplification or splitting but inhibited U2OS cell growth. G1 arrest and reduced EdU incorporation were observed in p150glued deficient U2OS cells. In addition, cyclin E was downregulated following p150glued depletion. The p53/p21 signaling was activated indicating that CDKs were inactivated. The reduced cell growth was ameliorated in the p150glued depleted cells when treated with p53 inhibitor. Thus, we have identified the centrosomal targeting of p150glued in distinct cell cycle stage and uncovered its role in controlling G1/S transition. J. Cell. Biochem. 116: 2049–2060, 2015. © 2015 Wiley Periodicals, Inc.