Objective Puerarin is a form of isoflavones obtained from Pueraria lobata. It stimulates hepatic metabolic function and lowers serum ALT, AST, and total-bilirubin level. The purpose of this study was to examine the effect of puerarin on nasopharyngeal carcinoma (NPC) CNE1 cells and preliminarily explore its possible mechanism. Materials and Methods CCK8 method was used to detect the proliferation activity of puerarin on NPC CNE1 cells and IC50 was calculated. CNE1 cells were treated with 0 μmol/L puerarin (containing equal volume of DMSO solution) as control group and 1000 μmol/L puerarin (IC50 concentration) as experimental group. Colony formation assay, Scratch-wound test and Transwell invasion assay were used to detect the clone formation ability, migration and invasion ability of puerarin on CNE1 cells. Then, RNA Sequencing was used to detect the changes of differentially expressed genes (DEGs) and signaling pathways after puerarin was applied to CNE1 cells. Results The inhibitory effect of puerarin on the proliferation activity of CNE1 cells was enhanced with the increase of concentration, and IC50 was calculated as 1000 μmol/L. Compared with the control group, the treatment of CNE1 cells with 1000 μmol/L puerarin could inhibit the clone formation, migration and invasion of CNE1 cells (P<0.05). A total of 379 DEGs were found by RNA sequencing, including 295 down-regulated genes and 84 up-regulated genes (padj<0.05). The significant differences in biological functions of differentially expressed genes were mainly distributed in “negative regulation of growth”, “angiogenesis”, “regulation of peptidase activity”, “positive regulation of vasculature development”, “digestion”, “positive regulation of angiogenesis”, “negative regulation of peptidase activity”, “extracellular matrix” and “Golgi lumen” (padj<0.05). Conclusion Puerarin could inhibit the proliferation, migration and invasion of NPC CNE1 cells, and its mechanism might be related to the inhibition of angiogenesis and cell growth.