B cells use B-cell receptors (BCRs) to sense antigens that are usually presented on substrates with different stiffness. However, it is not known how substrate stiffness affects B-cell proliferation, class switch, and in vivo antibody responses. We addressed these questions using polydimethylsiloxane (PDMS) substrates with different stiffness (20 or 1100kPa). Live cell imaging experiments suggested that antigens on stiffer substrates more efficiently trigger the synaptic accumulation of BCR and phospho-Syk molecules compared with antigens on softer substrates. In vitro expansion of mouse primary B cells shows different preferences for substrate stiffness when stimulated by different expansion stimuli. LPS equally drives B-cell proliferation on stiffer or softer substrates. Anti-CD40 antibodies enhance B-cell proliferation on stiffer substrates, while antigens enhance B-cell proliferation on softer substrates through a mechanism involving the enhanced phosphorylation of PI3K, Akt, and FoxO1. In vitro class switch differentiation of B cells prefers softer substrates. Lastly, NP67-Ficoll on softer substrates accounted for an enhanced antibody response in vivo. Thus, substrate stiffness regulates B-cell activation, proliferation, class switch, and T cell independent antibody responses in vivo, suggesting its broad application in manipulating the fate of B cells in vitro and in vivo.