Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.The liver is the main organ involved in biotransformation and the main expression site of enzymes from the cytochrome P450 family (CYP450). These enzymes constitute a large and multifunctional protein family, involved in many reactions related to the metabolism of exogenous compounds like xenobiotics and carcinogens. They also play a crucial role in the inactivation and activation of drugs [9]. CYP450 are monooxygenases, and they catalyze the incorporation of the oxygen atom to the substrate molecule with the simultaneous formation of a water molecule [10][11][12]. There are two phases of the biotransformation of drugs and xenobiotics in the human body. CYP450 enzymes play an important role in the course of the first-phase reactions. In these reactions, the introduction or unveiling of the -OH, -COOH, -SH, or -NH 2 functional groups occurs, which in turn, leads to an increase in the hydrophilicity of the drug/xenobiotic and allows second-phase reactions and consequently, expulsion from the body [13].One of the most important CYP450 subfamilies is the CYP2C subfamily, which metabolizes about 20% of all clinically used drugs and several endogenous compounds. Four isoenzymes are distinguished within this subfamily: 2C8, 2C9, 2C18, and 2C19 [14][15][16]. Of all the isoenzymes from the CYP2C subfamily, the CYP2C9 isoform is the most strongly expressed. It represents 50% of the entire subfamily and metabolizes about 15% of clinically used drugs, for example, non-steroidal anti-inflammatory drugs and diuretics [17,18]. As CYP enzymes are involved in the metabolism of exogenous compounds, their dysfunction may lead to slower elimination of dr...