Background: Fractalkine/CX3CL1, a surface chemokine, binds to CX3CR1 expressed by different lymphocyte subsets. Since CX3CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX3CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood. Methodology/Principal Findings: We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX3CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX3CR1 but only germinal centre B cells were attracted by soluble CX3CL1 in a transwell assay. CX3CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX3CR1+ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX3CL1. ELISA assay showed that soluble CX3CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX3CL1 did not attract spleen B cells from wild type mice. OVA immunized CX3CR1-/- or CX3CL1-/- mice showed significantly decreased specific IgG production compared to wild type mice. Conclusion/Significance: We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX3CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.