Biotin affects gene expression at both the transcriptional and the posttranscriptional level; biotin metabolites might have biotin-like activities with regard to gene expression. Here, human hepatocarcinoma (HepG2) cells were used (i) to identify clusters of biotin-dependent genes, (ii) to determine whether the naturally occurring metabolite bisnorbiotin affects gene expression and (iii) to determine whether biotin and bisnorbiotin affect the expression of genes coding for ribosomal subunits and translation initiation factors. HepG2 cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L, control) and pharmacological (10 nmol/L) concentrations of biotin; a fourth treatment group consisted of cells cultured in biotin-deficient medium (0.025 nmol/L) supplemented with bisnorbiotin (0.225 nmol/L). Gene expression was quantified by using DNA microarrays and reverse transcriptase polymerase chain reaction. The expression of 1803 genes depended on biotin concentrations in culture media; the expression of 618 genes depended on bisnorbiotin. Biotin deficiency was associated with increased expression of a gene cluster encoding ribosomal subunits and eukaryotic translation initiation factor 5A; this effect was reversed by supplementation with biotin and bisnorbiotin. Additional prominent clusters of (bisnor)biotin-dependent genes included DNA-, RNA-, and nucleotide-binding proteins, consistent with a role for biotin in cell signaling and gene expression. Collectively, these data suggest that bisnorbiotin has biotin-like activities regarding gene expression, and that clusters of (bisnor)biotin-dependent genes include genes that play roles in translational activity.