The outer mitochondria membrane 40 homolog (TOMM40) is thought to be involved in the mitochondrial function and to influence the susceptibility for the development of AD. To determine whether TOMM40 rs157581 polymorphism is a plausible modulator of spontaneous brain activity in amnestic mild cognitive impairment (aMCI) patients, 46 aMCI subjects and 21 healthy controls were recruited and explored. Each individual was firstly genotyped for TOMM40 rs157581 and was further assessed by resting-state functional MRI to evaluate regional brain activity using amplitude low-frequency fluctuation analysis (ALFF). aMCI patients showed decreased ALFF in the left inferior frontal gyrus and insula, and increased ALFF in right posterior cingulate, lingual gyrus and calcarine sulcus. A significant difference in the interaction of "groups × genotypes" was observed in the bilateral superior frontal gyrus, bilateral lingual gyrus, right calcarine sulcus and left cerebellum. These results demonstrated a pattern of change in ALFF values, in which increased and subsequently decreased ALFF values in parallel with the progression of aMCI symptoms. The present study shows for the first time that TOMM40 rs157581 polymorphism may modulate regional spontaneous brain activity and related to the progression of aMCI.