CD33 is a susceptibility locus for late-onset Alzheimer's disease (AD). However, how the neural mechanism of CD33 affects cognition in the AD spectrum population remains unclear. We aimed to investigate the primary and interactive effects of the CD33 (rs3865444) genotype on brain function in patients with AD using global functional connectivity density (gFCD) mapping via resting-state functional magnetic resonance imaging. Furthermore, we used a conditional process analysis to identify the relationship among the CD33 genotype, gFCD, and cognition performance across the AD spectrum population. Compared to cognitively normal (CN) and mild cognitively impaired (MCI) subjects, patients with AD showed higher gFCD in the default mode network, and the CD33 genotype primarily influenced brain function in the fronto-striatal circuit. Importantly, an interaction between the CD33 genotype and AD was observed in the parahippocampal gyrus. During disease progression, the gFCD trajectories of the CD33 A + allele gradually decreased, whereas those of the CD33 CC allele displayed an inverted U-shaped curve. Furthermore, gFCD in the dorsal anterior cingulate cortex positively mediated the relationship between the CD33 genotype and cognition, while gFCD in the precuneus bidirectionally moderated the mediation in the AD spectrum. These findings provide new insights into the neural mechanisms underlying the influence of the CD33 genotype on cognitive performance and highlight the importance of precise therapeutic strategies for high-risk AD populations.