INTRODUCTION: We previously demonstrated that regulating mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) affects axonal Aβ generation in a well-characterized three-dimensional (3D) neural Alzheimer's disease (AD) model. MAMs vary in thickness and length, impacting their functions. Here, we examined the effect of MAM thickness on Aβ in our 3D neural model of AD. METHODS: We employed fluorescence resonance energy transfer (FRET) or fluorescence-based MAM stabilizers, electron microscopy, Aβ enzyme-linked immunosorbent assay (ELISA), and live-cell imaging with kymography to assess how stabilizing MAMs of different gap widths influence Aβ production and MAM axonal mobility. RESULTS: Stabilizing tight MAMs (∼6 nm gap width) significantly increased Aβ levels, whereas basal (∼25 nm) and loose MAMs (∼40 nm) maintained or reduced Aβ levels, respectively. Tight MAMs reduced mitochondrial axonal velocity compared to basal MAMs, while loose MAMs showed severely reduced axonal distribution. DISCUSSION: Our findings suggest that stabilizing MAMs of specific gap widths, particularly in axons, without complete destabilization could be an effective therapeutic strategy for AD. HIGHLIGHTS: The stabilization of MAMs exacerbates or ameliorates Aβ generation from AD neurons in a MAM gap width-dependent manner. A specific stabilization threshold within the MAM gap width spectrum shifts the amyloidogenic process to non-amyloidogenic. Tight MAMs slow down mitochondrial axonal transport compared to lose MAMs offering a quantitative method for measuring MAM stabilization.