<p>Amelogenin (AMELX), the predominant extracellular matrix protein (EMP) in forming enamel, has a single phosphorylation site at Serine 16 (S16). Previously, we demonstrated that AMELX phosphorylation enhances its ability to stabilize amorphous calcium phosphate and inhibit mineralization. To study the role of AMELX phosphorylation in vivo, we substituted S16 with Ala to prevent phosphorylation in a generated <i>Amelx</i>^<i>S16A</i> knock-in (KI) mice. These KI mice exhibited hypoplastic enamel with ectopic calcifications, a lack of enamel rods, an accelerated transformation of amorphous calcium phosphate (ACP) to apatitic enamel crystals, and progressive pathological changes in ameloblasts, the enamel forming cells responsible for enamel production. The goal of this study was to test the hypothesis that the phosphorylation status of AMELX affects its interactions with other proteins in the extracellular enamel space. To determine the effect of AMELX phosphorylation on the protein–protein interactions, we have conducted Proximity Ligation Assays (PLA) of wild type (WT) and KI mandibular incisors. Specifically, we conducted PLA of Amelx with a transmembrane acid phosphatase 4 (Acp4), basal lamina protein laminin β3 (Lamβ3) and the EMP ameloblastin (Ambn). Quantitative image analysis showed significantly higher PLA signal for Acp4 in the ameloblast cell bodies and distal ends of WT mice. PLA signal was also significantly higher for Ambn in the ameloblast distal ends and in the enamel matrix of WT mice. For Lam5, the signal was also higher in ameloblasts cell bodies, ameloblast distal ends and the enamel matrix of WT mice. Collectively, the significantly higher PLA signals between phosphorylated Amelx and Ambn, Acp4, and Lamβ3, compared with nonphosphorylated Amelx, suggest that AMELX phosphorylation promotes potentially important protein–protein interactions during amelogenesis.</p>