The complexity of canonical Wnt signaling comes not only from the numerous components, but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagate signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1 or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all the three time points. Totally 287 proteins showed more than (including) 1.5-fold change in at least one time point. In addition to many known Wnt signaling transducers, more other phosphoproteins have been identified and quantitated, implicating their involvement in canonical Wnt signaling. K-means clustering analysis shows dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover, 100 unique phosphorylation sites were identified, with 54 of them quantitated in the three time points. Site-specific phosphopeptide quantitation revealed Ser20 phosphorylation on RRM2 increased upon 30 minutes’ Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNAi indicated that RRM2 functions downstream of -catenin as an inhibitor of Wnt signaling and Ser20 phosphorylation of RRM2 counteracts its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presents a comprehensive phosphorylation network regulated by canonical Wnt signaling, but also found novel molecules and phosphorylation involved in Wnt signaling.