MicroRNA-155 (miR-155) is expressed by cells of the immune system following activation and has been shown to be required for antibody production following vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and independent antigens. B cells lacking miR-155 generated reduced extra-follicular and germinal center responses and failed to produce high affinity IgG1 antibodies. Gene expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function-many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155 mediated inhibition. When Pu.1 is over-expressed in wild type B cells fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155 deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.