Traditional Chinese Medicine (TCM) has been served as complementary medicine for Ankylosing Spondylitis (AS) treatment for a long time. Yun-Pi-Yi-Shen-Tong-Du-Tang (Y-Y-T) is a novel empirical formula designed by Prof. Chengping Wen. In this study, a retrospective investigation supported efficacy of Y-Y-T and then we deciphered the underlying molecular mechanism of the efficacy. Herbal ingredients and targeting proteins were collected from TCMID. PPI networks were constructed to further infer the relationship among Y-Y-T, drugs used for treating AS, differentially expressed genes of AS patients and AS disease proteins. Finally, it was suggested that TLR signaling pathway and T cell receptor signaling pathway may involve in the biological processes of AS progression and contribute to the curative effect and proteins such as JAK2, STAT3, HSP90AA1, TNF and PTEN were the key targets. Our systemic investigation to infer therapeutic mechanism of Y-Y-T for AS treatment provides a new insight in understanding TCM pharmacology.