MicroRNAs (miRNAs) post-transcriptionally regulate mRNA expression, controlling global cell function. Altered expression or function of miRNAs causes various diseases. Chemically induced changes in miRNA expression in human tissues are not fully understood. We investigated the changes in miRNA expression by rifampicin, which modulates the expression of various genes related to drug metabolism and pharmacokinetics, in human hepatocytes, and evaluated the relationship with the gene expression changes. We found that 23 miRNAs were increased (>2-fold) and 17 miRNAs were decreased (<0.5-fold) among 150 detected miRNAs, whereas 60 genes were increased and 105 genes were decreased among 22,673 detected genes upon treatment with 10 µM rifampicin. Changes in 17 intragenic miRNAs out of 40 altered miRNAs did not occur in parallel with alterations in their host genes. We searched for the target mRNAs of the miRNAs altered by rifampicin and found that the changes in expression of 16 mRNA/miRNA pairs were inversely associated. Thus, some mRNA expression altered by rifampicin may result from miRNA regulation. In conclusion, we found that rifampicin altered miRNA expression in human hepatocytes. We obtained new insight on the mechanism of the miRNA expression changes and the complicated relationship with gene transcripts.