Recent evidence has suggested microRNAs as viable therapeutic targets for a wide range of human disease. However, lack of gene‐specificity of microRNA actions may hinder this application. Here we developed two new approaches, the gene‐specific microRNA mimic and microRNA‐masking antisense approaches, to explore the possibility of using microRNA's principle of actions in a gene‐specific manner. We examined the value of these strategies as rational approaches to develop heart rate‐reducing agents and “biological pacemakers” by manipulating the expression of the cardiac pacemaker channel genes HCN2 and HCN4. We showed that the gene‐specific microRNA mimics, 22‐nt RNAs designed to target the 3′untranslated regions (3′UTRs) of HCN2 and HCN4, respectively, were efficient in abrogating expression and function of HCN2 and HCN4. The gene‐specific microRNA mimics repressed protein levels, accompanied by depressed f‐channel conductance and the associated rhythmic activity, without affecting mRNA levels of HCN2 and HCN4. Meanwhile, we also designed the microRNA‐masking antisense based on the miR‐1 and miR‐133 target sites in the 3′UTRs of HCN2 and HCN4 and found that these antisense oligodeoxynucleotides markedly enhanced HCN2/HCN4 expression and function, as reflected by increased protein levels of HCN2/HCN4 and I_f conductance, by removing the repression of HCN2/HCN4 expression induced by endogenous miR‐1/miR‐133. The experimental examination of these techniques and the resultant findings not only indicate feasibility of interfering miRNA action in a gene‐specific fashion but also may provide a new research tool for studying function of miRNAs. The new approaches also have the potential of becoming alternative gene therapy strategies. J. Cell. Physiol. 212: 285–292, 2007. © 2007 Wiley‐Liss, Inc.