A non-cognate mechanism of protection against human immunodeficiency virus-1 (HIV-1) infection involves up-regulation of β-chemokines, which bind and may down-modulate the CCR5 co-receptors, thereby preventing transmission of M-tropic HIV-1. The objective of this investigation was to evaluate this mechanism in vivo in non-human primates. Rhesus macaques were immunized by a modified targeted lymph nodes (TLN) route with recombinant simian immunodeficiency virus (SIV) glycoprotein 120 (gp120) and p27 in alum, and adsorbed recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) with either interleukin (IL)-2 or IL-4. Immunization induced significant increases in the concentrations of CD8 cell-derived suppressor factor (CD8-SF), regulated on activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α and MIP-1β, and down-modulation of the proportion of cells expressing CCR5 (r = 0.737, P < 0.05). The macaques were then challenged with SIVmac 220 by the rectal mucosal route. The plasma SIVmac RNA showed a significant inverse correlation with the CD8-SF or the concentration of the three β-chemokines (r = 0.831 and 0.824, P < 0.01), but a positive correlation between the proportion of CCR5+ cells and SIVmac RNA (r = 0.613, P = 0.05). These results demonstrate for the first time in vivo that immunization up-regulates β-chemokines, which may down-modulate CCR5 co-receptors, and both functions are significantly correlated with the viral load. Hence, the non-cognate β-chemokine-CCR5 mechanism should be considered as complementary to specific immunity in vaccination against HIV.