American ginseng (Panax quinquefolius L., Araliaceae) possesses anti-cancer potential and is one of the most commonly used herbal medicines in the United States. Ginsenoside Rg3, one of the saponins in American ginseng, has been shown to inhibit tumor growth. In this study, we sought to characterize the downstream genes targeted by American ginseng extracts in HCT-116 human colorectal cancer cells. We first demonstrated that the content of Rg3 in American ginseng steamed at 120°C for 2 h (referred to as S2h) was significantly increased when compared with that of the unsteamed ginseng. Both S2h and Rg3 exhibited anti-proliferative effects on HCT-116 cells. Using the Affymetrix high density genechips containing more than 40,000 genes and ESTs, the gene expression profiling of HCT-116 cells were assayed. Microarray data indicated that the expression levels of 76 genes were changed significantly after treatment with S2h or Rg3, whereby it was found that 52 of the 76 genes were up-regulated while the remaining 24 were down-regulated. Ingenuity Pathways Analysis of top functions affected by both S2h and Rg3 were carried out. The most effected pathway is the Ephrin receptor pathway. To validate the microarray data, quantitative real-time PCR of six candidate target genes was conducted, whereby it was found that three genes were up-regulated (AKAPA8L, PMPCB and PDE5A) and three were down-regulated (PITPNA, DUS2L and RIC8A). Although further studies are needed to elucidate the mechanisms of action, our findings should expand the understanding of the molecular framework of American ginseng as an anti-cancer agent.