Background: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. Methods: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcus pyogenes proteomes were used to identify mimic epitopes. Results: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8 ⁺ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8 ⁺ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8 ⁺ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8 ⁺ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and S pyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8 ⁺ T-cell activation in vitro. Conclusions: ProTα-dependent CD8 ⁺ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8 ⁺ T cells, suggesting mechanisms provoking autoimmunity.