Abstract Endoplasmic reticulum (ER) calcium (Ca 2+ ) homeostasis is essential for maintaining normal cellular physiological functions. Its disturbance is strongly linked to the onset and progression of human diseases, including cancer, developmental defects, and neurodegenerative disorders. The lack of sensitive ratiometric ER Ca 2+ indicators, nevertheless, hinders systematic investigation of ER Ca 2+ modulators and the underlying mechanisms. Capitalizing on two ultra-sensitive ER Ca 2+ indicators and CRISPR-based genome-wide screening, we identified a set of proteins capable of reducing the ER Ca 2+ content. Further comparative analysis and qPCR validation pinpointed adenylate cyclase 9 (AC9), which is upregulated during neuronal differentiation, as a key ER-Ca 2+ -reducing regulator. Mechanistically, AC9-mediated production of cAMP is not essential for its ability to reduce ER Ca 2+ content. Instead, AC9 inhibits store operated calcium entry (SOCE) by acting on Orai1, ultimately causing attenuation of ER Ca 2+ level. More physiologically relevant, upregulation of AC9 in neurons is essential for reducing ER Ca 2+ levels during Drosophila brain development. Collectively, this study lays a solid groundwork for further in-depth exploration of the regulatory mechanisms dictating ER Ca 2+ homeostasis during neuronal differentiation and brain development.