In this study, we show that IFNα increases the chemotaxis of human B cells to CCL20, CCL21 and CXCL12 in a dose- and time-dependent manner. The effect was maximal with 2000 IU ml⁻¹ IFNα. It peaked at 24 h and decreased thereafter. At 24 h, IFNα had increased B-cell chemotaxis to CCL20 by 20 ± 6.2% (n = 9, P < 0.002), to CCL21 by 20 ± 8.5% (n = 14, P < 0.0001) and to CXCL12 by 16.3 ± 4.2% (n = 12, P < 0.003) without changing CCR6, CCR7 or CXCR4 expression. IFNα enhanced the migration of memory B cells to CCL20, CCL21 and CXCL12 2.6-fold more strongly than that of naive B cells. The triggering of chemokine receptors by their ligands resulted in the activation of phosphatidylinositide-3 kinase (PI3K)/protein kinase B (PKB), inhibitory NF-κB (IκBα) RhoA and extracellular signal-regulated protein kinase 1/2 (ERK1/2). All these effectors except ERK1/2 are crucial for B-cell chemotaxis. IFNα modulated the requirements for B-cell chemotaxis, which became dependent on ERK1/2, more dependent on PI3K, RhoA and nuclear factor-κB but less dependent on Gβγ and phospholipase C activation. IFNα also decreased ligand-induced chemokine receptor internalization in a manner dependent on PI3K/AKT and RhoA but not on IκBα and ERK1/2. Our data characterize chemokine receptor signaling in human B cells and clarify the relevance of downstream pathways in B-cell chemotaxis and chemokine receptor internalization. They also suggest that non-class I PI3K are involved in B-cell chemotaxis.