We describe the efficient use of artificial microRNAs (amiRs) in Marchantia polymorpha using both endogenous and heterologous primary microRNA (pri-miR) hairpin backbones. Targeting of two transcription factor genes, MpARF1 and MpRR-B, mediating different hormonal responses, demonstrated that amiRs can create specific and reproducible physiological and morphological defects, facilitating interpretation of gene function. A third amiR was designed to target a gene encoding a component of the Polycomb recessive complex 2, MpE(z), and constitutive expression of this amiR results in sporeling lethality. Adaptation of an estrogen-inducible system allowed analysis of the phenotypic effects of induction of this amiR during other stages of the life cycle. We discuss the advantages and challenges of the use of amiRs as a tool for reverse genetic analysis in M. polymorpha.,© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.