The ATP-sensitive K⁺ (K_ATP) channel, composed of Kir6.2 and sulfonylurea receptor (SUR1), in pancreatic β-cells is believed to serve as a metabolic sensor regulating insulin secretion according to glucose levels. Thus, genetic disruption of Kir6.2 expression may impair K_ATP channel function in glucose sensing and insulin secretion. Here we show evidence obtained from functional genetic assays supporting this hypothesis. To avoid adaptive cellular mechanisms in transgenic preparations, we designed a hammerhead ribozyme that specifically targeted the Kir6.2 mRNA at serine 78. The Kir6.2-ribozyme was constructed in an adenoviral vector and expressed in insulin-secreting RINm5F cells. Both RT-PCR and Northern blot analyses showed that Kir6.2 transcripts were significantly reduced with a Kir6.2-ribozyme treatment. Whole-cell patch-clamp studies indicated that the Kir6.2-ribozyme treatment lowered K_ATP channel density by 66%. In response to higher glucose challenge, insulin release from the RINm5F cells dropped by approximately 20% in a transfection dose of 0.7 multiplicity of infection, and by 30–40% in a dose of 2.7 multiplicity of infection. These results therefore indicate that K_ATP channels play an important role in glucose sensing and insulin secretion, and ribozyme Kir6.2-gene targeting is an effective approach for selective inhibition of functional expression of K_ATP channels.