Arf proteins are small GTPases that function as molecular switches and cycle between their active (GTP) and inactive (GDP) forms. Their activation is mediated by guanine nucleotide exchange factors (GEFs), and their inactivation is facilitated by GTPase-activating proteins (GAPs). Since we have previously shown that murine cytomegalovirus (MCMV) infection triggers massive reorganization of the Arf system early in infection, the aim of this study was to determine the contribution of Arf proteins in the establishment of MCMV infection. Balb 3T3 murine fibroblasts with knockdown of Arf protein expression by small interfering RNAs (siRNAs) were infected with MCMV and the expression of immediate early (IE1) and early (E1, m06, and m25) viral proteins was followed by Western blot and immunofluorescence microscopy. In addition, cells were infected with C3X-GFP MCMV, in which a GFP expression cassette was inserted upstream of the ie2 gene, and the intensity of green fluorescence was monitored by flow cytometry as an indicator for progression of the immediate-early phase of infection. The expression of IE1, E1, m25, and m06 proteins were drastically reduced in cells transfected with siArf1 or siArf6, in contrast to cells transfected with siArf3, siArf4, or siArf5, in which the expression of the viral proteins was similar to that in control cells. In C3X-GFP MCMV infected cells, the green fluorescence intensity was significantly decreased in siArf1- and siArf6-transfected cells compared with the fluorescence intensity of control cells, suggesting that depletion of Arf1 and Arf6 prevents entry into the immediate–early phase of MCMV gene expression. Our results suggest that Arf1 and Arf6 are required in the earliest stages of MCMV infection before the MCMV genome enters the nucleus.