Activation of the canonical Wnt signalling pathway results in stabilisation and nuclear translocation of β-catenin. In the absence of a Wnt signal, β-catenin is phosphorylated at four conserved serine and threonine residues at the N-terminus of the protein, which results in β-catenin ubiquitination and proteasome-dependent degradation. The phosphorylation of three of these residues, Thr41, Ser37, and Ser33, is mediated by glycogen synthase kinase-3 (GSK-3) in a sequential manner, beginning from the C-terminal Thr41. It has recently been shown that the GSK-3 dependent phosphorylation of β-catenin requires prior priming through phosphorylation of Ser45. However, it is not known whether phosphorylation of Ser45 is carried out by GSK-3 itself or by an alternative kinase. In this study, the phosphorylation of β-catenin at Ser45 was characterised using a phospho-specific antibody. GSK-3β was found to be unable to phosphorylate β-catenin at Ser45 in vitro and in intact cells. However, inhibition of GSK-3 in intact cells reduced Ser45 phosphorylation, suggesting that GSK-3 kinase activity is required for the phosphorylation event. In vitro, CK1, but not CK2, phosphorylates Ser45. Ser45 phosphorylation in intact cells is not mediated by CK1ε, a known positive regulator of Wnt signalling, as overexpression of this kinase leads to decreased phosphorylation levels. In conclusion, phosphorylation of β-catenin at the GSK-3 priming site Ser45 is not mediated by GSK-3 itself, but by an alternative kinase, indicating that β-catenin is not an unprimed substrate for GSK-3 in vivo. Priming of GSK-3 dependent phosphorylation of β-catenin by a different kinase could have important implications for the regulation of Wnt signalling.