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Introduction: Alzheimer’s disease (AD) is an age-related disorder, characterized by the gradual loss of memory and cognitive function owing to neuronal damage and brain shrinkage. This study aimed to investigate how intranasal injection of human adiposederived stem cell-conditioned media (hADSC-CM) ameliorates cognitive performance and affects the level of estrogen receptor beta (Erβ) in the hippocampus of rats in an AD model. Methods: A total of 32 male rats were divided into four groups, including the control, AD model, hADSC-CM, and vehicle groups. The Morris water maze was used to assess the animals’ behavioral changes. Moreover, Nissl and Thioflavin-S staining were performed to evaluate the histology of the hippocampus. Immunohistochemistry was also carried out to evaluate the expression level of Erβ. Results: The intranasal injection of hADSC-CM improved the rats’ cognitive performance by reducing the number of dark cells and beta-amyloid plaques in the hippocampus in the AD model. Besides, the intranasal injection of hADSC-CM increased the level of Erβ in this model. Conclusion: The present findings indicated that the intranasal injection of hADSC-CM ameliorated cognitive function. Amyloid plaques and dark cells also diminished in the CA1 area of the hippocampus. Moreover, the expression level of ERβ increased. It can be concluded that hADSC-CM has significant treatment benefits for AD in rats. [ABSTRACT FROM AUTHOR]