Alzheimer's disease (AD) is a progressive neurodegenerative condition characterized by extracellular accumulation of amyloid-beta. Estrogen receptors (ERs) are widely recognized as mediators of neuroprotection from various insults. Recent research has highlighted the distinction between nuclear and non-nuclear ER activation mechanisms, positioning non-nuclear ERs as a safer option, devoid of the cardiovascular and cancer risks associated with nuclear ER activation. In this study, we aimed to confirm the neuroprotective potential of PaPE-1, a novel compound that preferentially activates non-nuclear ER signaling pathways, and to reveal its impact on autophagy, a process crucial in brain homeostasis and often deficient in AD. To model AD, we employed mouse primary neocortical cell cultures exposed to amyloid-beta oligomers. Treatment with PaPE-1 began after 24h of exposure to amyloid-beta and lasted for 6 h. We assessed the intensity of neuron-specific MAP2 immunofluorescent staining, the expression of AD-related markers (App, Bace1, Bace2, Mapt, Rcan, Ide), and autophagy (autophagosome formation, DNA methylation, mRNA expression, and protein levels of BECN1/Becn1 and ATG7/Atg7). Amyloid-beta decreased MAP2 staining intensity and upregulated App and Bace2, while treatment with PaPE-1 reversed these changes. In addition, PaPE-1 counteracted amyloid-beta-induced autophagy impairment as shown by the increased number of autophagosomes and DNA methylation, mRNA expression, and/or protein level changes. This study confirmed the neuroprotective potential of PaPE-1 and revealed its novel mechanisms of action, giving additional evidence that targeting non-nuclear ER signaling pathways is a promising approach in novel AD therapies. Funding: National Science Centre of Poland, grant number 2020/39/NZ7/00974