Mammalian AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status. It is activated by phosphorylation of the catalytic subunit on Thr172. The main objective of this study was the identification of a phosphatase involved in the regulation of AMPK activity. Mouse MIN6 β cells were used to study the glucose-induced regulation of the phosphorylation of AMPK. Small interfering RNA (siRNA) technology was used to deplete putative phosphatase candidate genes that could affect AMPK regulation. The effect of the siRNAs used in the study was compared with the effect observed using a negative control siRNA. A protein phosphatase complex composed of the catalytic subunit of protein phosphatase-1 (PP1) and the regulatory subunit R6 participates in the glucose-induced dephosphorylation of AMPK. R6 interacts physically with the β-subunit of the AMPK complex and recruits PP1 to dephosphorylate the catalytic α-subunit on Thr172. siRNA depletion of R6 decreases glucose-induced insulin secretion due to the presence of a constitutively active AMPK complex. The characterization of the PP1-R6 complex identifies this holoenzyme as a possible target for therapeutic intervention with the aim of regulating the activity of AMPK in pancreatic β cells.