Early infection with Leishmania donovani during visceral leishmaniasis (VL) results in the enhanced expression of C-C chemokine receptor 5 (CCR5) in macrophages. CCR5 expression at different time points of infection revealed increased expression at protein and mRNA level from 3 to 24 h and beyond 24 h expression of CCR5 is downregulated. To better understand the functional role of CCR5 during leishmania infection, RNA interference strategy has been used to modulate macrophage function by targeting the expression of CCR5 gene. We found that silencing of CCR5 receptor expression by transfection of CCR5-specific small interfering RNA (siRNA) in murine peritoneal macrophages restricted the parasitic burden up to 70% during early hours of infection. In addition, gene silencing of CCR5 prior to L. donovani infection enhanced the pro-inflammatory response of the host macrophages in comparison with infection alone as shown by high nitric oxide generation and the TNF-alpha:IL-10 ratio. These findings suggest that CCR5 receptor plays a significant role in the entry and establishment of L. donovani in murine macrophages and CCR5 gene silencing would be a potent therapeutic approach to control VL by restricting parasite entry.