Protein kinase Calpha (PKCalpha) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCalpha in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCalpha to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCalpha knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCalpha membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCalpha membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCalpha with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCalpha. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCalpha even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCalpha scaffold to promote PKCalpha-mediated EGFR inactivation through increased membrane targeting of PKCalpha and EGFR/PKCalpha complex formation.Oncogene advance online publication, 16 July 2012; doi:10.1038/onc.2012.303.