: Purpose: To use label-free proteomic method to investigate the mechanism of action of nuanggong zhitong decoction (NZD) on primary dysmenorrhea (PD). Methods: A mouse model of PD was established through oxytocin administration. The mice were divided into control group (normal mice), model group (PD mice administered normal saline), and treatment group (mice given NZD). The serum levels of PGE2 and PGF2α in the mice were measured by ELISA. The differentially expressed proteins (DEPs) among the three groups were revealed by identifying the proteins that were up-regulated (or down-regulated) in model group and down-regulated (or up-regulated) in the treatment group. The DEPs in the three groups were identified using NanoHPLC-MS/MS, and their functions were investigated using bioinformatics analyses. The accuracy of proteomics was verified with western blot analysis. Results: Thirty-eight up-regulated and 66 down-regulated DEPs were identified. Bioinformatics analysis revealed that the DEPs were related to immune response, signal conduction, protein binding, and metabolism. STRING analysis indicated a total of 53 DEPs have direct or indirect functional links. Western blot results revealed that levels of Stat1, Rock1, vinculin and vaveolin-1 were consistent with the results of proteomic analysis. Conclusion: These findings provide further insights into the mechanism underlying the protective effects of NZD.