Oil-in-water emulsions are potent human adjuvants used for effective pandemic influenza vaccines; however, their mechanism of action is still unknown. By combining microarray and immunofluorescence analysis, we monitored the effects of the adjuvants MF59 oil-in-water emulsion, CpG, and alum in the mouse muscle. MF59 induced a time-dependent change in the expression of 891 genes, whereas CpG and alum regulated 387 and 312 genes, respectively. All adjuvants modulated a common set of 168 genes and promoted antigen-presenting cell recruitment. MF59 was the stronger inducer of cytokines, cytokine receptors, adhesion molecules involved in leukocyte migration, and antigen-presentation genes. In addition, MF59 triggered a more rapid influx of CD11b+ blood cells compared with other adjuvants. The early biomarkers selected by microarray, JunB and Ptx3, were used to identify skeletal muscle as a direct target of MF59. We propose that oil-in-water emulsions are the most efficient human vaccine adjuvants, because they induce an early and strong immunocompetent environment at the injection site by targeting muscle cells.