ABSTRACT Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody‐mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin‐β‐receptor (LTβR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal‐center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTβR‐Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs . 2.2 in control recipients; P =0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTβR‐Ig (median survival time: 18 vs . >50 d, respectively, P =0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.—Motallebzadeh, R., Rehakova, S., Conlon, T. M., Win, T. S., Callaghan, C. J., Goddard, M., Bolton, E. M., Ruddle, N. H., Bradley, J. A., Pettigrew, G. J. Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses. FASEB J. 26, 51–62 (2012). www.fasebj.org