Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway,Ins1(insulin 1),Ins2(insulin 2),Kcnj11(potassium inwardly-rectifying channel, subfamily J, member 11),Kcnn1(potassium calcium-activated channel subfamily N member 1),Rab3a(member RAS oncogene family),Gnas(GNAS complex locus),Syt13(synaptotagmin 13),Pax6(paired box 6),Klf11(Kruppel-Like Factor 11), andNr4a1(nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, whileIns1andIns2transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6(nuclear receptor coactivator 6),Pax6,Kcnj11,Syt13,Stxbp1(syntaxin binding protein 1), andSnap25(synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells.