This study aimed to determine the expression and potential interaction of 5-HT4 and 5-HT6 receptors in HEK293 and SH-SY5Y cell lines, to specify the role of N-linked glycosylation of the 5-HT4 receptor, and to assess the expression of these receptors at different stages of Alzheimer's disease (AD). The results showed that both the cell lines natively express the 5-HT4 receptors but at a low level which prevented the detection of ERK1/2 phosphorylation. Contrastingly, overexpression of these receptors individually in HEK293 cells showed transient ERK1/2 phosphorylation, while concurrent overexpression of the receptors did not show augmentation effects in pERK1/2 level following 5-HT stimulation. The overexpressed 5-HT4 receptor showed the presence of N-glycosylation. Abrogation of asparagine at 180 position reduced the apparent weight and cell surface expression of 5-HT4 receptor. Moreover, the 5-HT4 receptor was significantly downregulated in limbic and neocortical stages of AD relative to controls, whereas the 5-HT6 receptor expression was reduced in the neocortical stage relative to the limbic stage of AD. The variations in the receptor expression significantly correlated with the cognitive status of AD patients besides the phospho-tau. These data suggest that the early modulation of these receptors might improve AD and its associated symptoms.