The dominant-negative inhibition of KCNJ2-encoded inward rectifier potassium channels (Kir2) is currently considered the best approach to biological pacemakers. We hypothesized that inhibition of the inward rectifier potassium current (IK1) in ventricular myocytes by RNA interference (RNAi) would convert ventricular myocytes into pacemaker cells. Five pieces of short hairpin RNA (shRNA) were designed to target the KCNJ2 gene and then plasmids incorporating shRNA and green fluorescent protein (GFP) as a marker were constructed for transfection into rat ventricular myocytes. The levels of KCNJ2 mRNA were analyzed with real-time quantitative RT-PCR to screen for pieces of shRNA that were effective at inhibiting the expression of the KCNJ2 gene. The activity of potassium ionic channels was then studied in the transfected ventricular myocytes. In the recombinant plasmids, LYS2 transfection significantly inhibited the mRNA of the KCNJ2 gene in comparison to other groups (p < 0.05), and the beating frequency of ventricular myocytes increased after LYS2 transfection. The open probability of IK1 potassium ion channels of cardiac myocytes transfected with the LYS2 plasmid was significantly downregulated (p < 0.05) and the IK1 of ventricular myocytes was also significantly suppressed compared to the negative group (p < 0.05). Our study demonstrated that IK1 was clearly inhibited after the inhibition of KCNJ2 gene expression by RNAi, and this may represent a new approach to the study of biological pacemakers.