Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca²⁺ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca²⁺ channels IP₃R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca²⁺ pump SERCA2 (Sarco/endoplasmic reticulum Ca²⁺ ATPase 2) and the ER Ca²⁺ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca²⁺ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca²⁺ from the ER upon carbachol-induced Ca²⁺ release through IP₃Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca²⁺ signaling.