Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiological in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et. al. 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long term human hepatocyte culture model (HepatoPac®) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by siRNA with subsequent recovery, as well as up regulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UGT2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings.