Age-related cognitive impairment, for instance Alzheimer’s disease (AD), is a chronic, progressive, neurodegeneration disease. The concentration of endogenous formaldehyde (FA) positively correlates with the severity of cognitive impairments in AD patients. However, the FA concentrations used in the previous studies were usually higher than the physiological and pathological levels in aging people. To elucidate the relationship between FA and the pathogenesis of AD, it is necessary to investigate the effect of long-term exposure of neurons to low concentration of FA, which is consistent with the pathological FA concentration. In this study, we established a cell culture method to simulate the chronic low-concentration FA exposure by using a serial passage strategy. Murine neuroblastoma N2a cells and primary murine hippocampal neurons were exposed to a simulated pathological FA concentration referred to AD patients. During the long-term of culture, FA gradually accumulated in the medium and impaired N2a cells. High performance liquid chromatography, cell viability assay and lactate dehydrogenase assay showed that, the FA-elimination capacity of N2a cell decreases with the incubation time, accompanied with inhibition of cell growth and increase in cell death. Holographic microscopy showed that long-term simulated pathological FA exposure attenuated the cells’ adhesive morphology. Cells exposed to FA became thicker, exhibiting impairment of neuronal processes. The number of primary neurites in primary hippocampal neurons were reduced by FA exposure, suggesting a decrease in the connectivity between neurons. Formaldehyde accumulation promoted Tau phosphorylation at its Thr181 and Ser396 epitopes, which may be one of the factors leading to decrease in primary neurites. Our findings indicate that accumulation of simulated pathological concentration of FA impairs neurons, induces Tau hyperphosphrylation and decreases neural connectivity, which would lead to neural dysfunction and eventually contribute to the pathogenesis of age-related cognitive impairment.