Alzheimer disease (AD) is pathologically characterized by the deposition of the amyloid-β peptide (Aβ) as senile plaques in the brain. Aβ is generated through the sequential cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Whereas the aberrant metabolism of Aβ has been implicated in the etiology of AD, the precise regulatory mechanism of Aβ generation remains unclear. In this study, we established a genetic screening based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. And we successfully identified calcium and integrin-binding protein 1 (CIB1) as a negative regulator of Aβ production. CIB1 is a ubiquitously expressed, myristoylated, calcium-binding adaptor protein. We found that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.