MicroRNAs (miRNAs) are non-coding RNAs that function as guide molecules in gene silencing pathways. This study provides the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). Primary miRNA transcripts (pri-miRNAs) contain cap structures and poly(A) tails, unique features of class II gene transcripts. Treatment of human cells with α-amanitin, which inhibits pol II activity, reduced pri-miRNA levels, indicating that pol II is involved in miRNA transcription. Chromatin immunoprecipitation analyses showed that pol II is physically associated with miRNA promoters. The study also determined the promoter and terminator of the miR-23a~27a~24-2 gene, confirming that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. These findings suggest that miRNA genes are transcribed by pol II and share characteristics with class II genes. The study provides insights into the structure and regulation of miRNA genes, highlighting the importance of understanding miRNA biogenesis and its regulatory mechanisms.MicroRNAs (miRNAs) are non-coding RNAs that function as guide molecules in gene silencing pathways. This study provides the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). Primary miRNA transcripts (pri-miRNAs) contain cap structures and poly(A) tails, unique features of class II gene transcripts. Treatment of human cells with α-amanitin, which inhibits pol II activity, reduced pri-miRNA levels, indicating that pol II is involved in miRNA transcription. Chromatin immunoprecipitation analyses showed that pol II is physically associated with miRNA promoters. The study also determined the promoter and terminator of the miR-23a~27a~24-2 gene, confirming that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. These findings suggest that miRNA genes are transcribed by pol II and share characteristics with class II genes. The study provides insights into the structure and regulation of miRNA genes, highlighting the importance of understanding miRNA biogenesis and its regulatory mechanisms.