Background: Tumor cell death in response to an immunotherapy may lead to the release of secondary (i.e. non-targeted) tumor antigens that prime subsequent immune responses (antigen spread). This may contribute to tumor control and reveal post-treatment biomarkers of clinical outcome. Sipuleucel-T, approved in the U.S. for treatment of patients with asymptomatic or minimally symptomatic, metastatic castration-resistant prostate cancer (mCRPC), is designed to elicit an immune response to prostatic acid phosphatase. Here we show that sipuleucel-T also elicits humoral antigen spread that may be associated with clinical benefit. Methods: We used protein microarrays and Luminex xMAP to identify and validate post-treatment serum immunoglobulin G (IgG) responses against secondary antigens. Association between IgG responder status (≥ 2-fold IgG elevation post-treatment) and overall survival (OS) was assessed using a Cox regression model adjusted for baseline prognostic factors (prostate-specific antigen [PSA], lactate dehydrogenase [LDH], bone lesions, Gleason score) and prior bisphosphonate use. Results: In IMPACT (NCT00065442, the pivotal trial of sipuleucel-T), IgG responses against secondary antigens were consistently observed three to four months post-treatment in the sipuleucel-T arm but not in the control arm. In evaluable sera from IMPACT (sipuleucel-T n=93; control n=40), IgG responses against an array of secondary antigens, including an oncogene (K-RAS) and a prostate-specific antigen (KLK2/hK2), were confirmed by Luminex xMAP in sipuleucel-T-treated patients (Wilcoxon test, p≤10^-6); responses were not observed in control patients (p>0.1). Antigen spread was also observed post-sipuleucel-T treatment in ProACT (Wilcoxon test, p≤0.01), an independent study in patients with mCRPC (NCT00715078, evaluable n=26). In IMPACT, OS was longer in sipuleucel-T-treatment patients with IgG response to greater than or equal to two secondary antigens than in those without such response (Cox model, p≤0.01, HR≤0.4). Conclusions: Sipuleucel-T treatment resulted in humoral antigen spread that was associated with improved OS in IMPACT. These results contribute to an understanding of sipuleucel-T’s mechanism of action, and may enable identification of post-treatment biomarkers of clinical outcome. Clinical trial information: NCT00065442.