Alzheimer's disease is a worldwide metabolic disease and an economically costly disease to society, so more medicines need to be developed to treat this disease. Huperzine A, a novel lycopodium alkaloid isolated from traditional Chinese medicine Huperzia serrata (Qian Ceng Ta), has been shown to possess multiple neuroprotective effects for Alzheimer's disease, but the precise pharmacological mechanism of huperzine A is unclear and needs to be further investigated. In this study, proteins from untreated N2a cells (Con group), cells preincubated with huperzine A followed by Aβ (1–42) oligomers treatment (HupA group) and cells treated with Aβ (1–42) oligomers (Aβ group) with five biological replicates in each cohort, were processed in a centrifugal proteomic reactor and quantified by label-free quantitation. A total of 2860 proteins were quantified with high confidence, and 198 proteins were significantly changed (with p-value < 0.05) between HupA and Aβ cohorts. The pathway and direct protein–protein interaction network analysis showed that huperzine A protects N2a cells against Aβ oligomer-induced cell death by downregulation of cellular tumor antigen p53 (Trp53) expression.