A core component of the immune system are B cells, which are activated by infection and then mature to provide long-lived immunity. Activation is initiated when a cell surface B cell receptor, in association with its coreceptor, recognizes an antigen. Susa et al. report a structure of the B cell receptor CD81 in complex with its co receptor, CD19. CD81 alone binds to cholesterol, but the conformational changes associated with binding to CD19 occlude the cholesterol-binding pocket. Regulating cholesterol binding could play a role in the activation mechanism. The structure also provides a basis for the design of immunotherapies.Science, this issue p. 300Signaling through the CD19-CD81 co-receptor complex, in combination with the B cell receptor, is a critical determinant of B cell development and activation. It is unknown how CD81 engages CD19 to enable co-receptor function. Here, we report a 3.8-angstrom structure of the CD19-CD81 complex bound to a therapeutic antigen-binding fragment, determined by cryo–electron microscopy (cryo-EM). The structure includes both the extracellular domains and the transmembrane helices of the complex, revealing a contact interface between the ectodomains that drives complex formation. Upon binding to CD19, CD81 opens its ectodomain to expose a hydrophobic CD19-binding surface and reorganizes its transmembrane helices to occlude a cholesterol binding pocket present in the apoprotein. Our data reveal the structural basis for CD19-CD81 complex assembly, providing a foundation for rational design of therapies for B cell dysfunction.