Lymphoma is considered to be one of the most pressing health problems worldwide owing to its high incidence and mortality. Previous studies have shown that periplocin, a naturally occurring compound, inhibits growth and induces apoptosis in several cancers. However, the effects of periplocin on lymphoma and the underlying mechanisms of action remain unclear. The PharmMapper database was used to predict the potential targets of periplocin. The GeneCard database was used to identify lymphoma-related genes. A few intersecting genes were obtained, and the protein-protein interaction network was visualized using STRING Gene ontology analysis. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using R project. MTS assay, flow cytometry, real-time quantitative polymerase chain reaction (qPCR), and Western blotting were used to verify whether periplocin possesses anti-lymphoma activity. A total of 216 intersecting genes were identified. Numerous cancer-related signaling pathways were visualized using Cytoscape software, with the PI3K-Akt signaling pathway being the highest-ranked pathway related to cell proliferation, apoptosis, and cell cycle progression. HuT 78 and Jurkat cell lines were used to verify the predictions. Periplocin significantly inhibited their proliferation in a dose- and time-dependent manner, but had no effect on the viability of peripheral blood lymphocytes. Flow cytometry revealed that treatment with periplocin increased the apoptotic rate and ratio of HuT 78 and Jurkat cells in the G2/M phase. CDK1 and cyclin B1 complex formation is a key gatekeeper to mitotic division in the G2/M phase. Western blot analysis revealed that periplocin significantly decreased the protein levels of CDK1 and cyclin B1; however, real-time qPCR revealed no effect on gene expression. Periplocin showed anti-tumor effects in lymphoma cells through multiple targets and signaling pathways, and could be a novel therapeutic agent for the treatment of lymphoma. © 2021 Zhao et al.