Dysregulation of gluconeogenesis contributes to the pathogenesis of metabolic disease, such as type-2 diabetes. The role of long non-coding RNAs (IncRNAs) in the pathogenesis of diabetes has recently received increased attention. In the present study, we identified a novel IncRNA, betaine-homocysteine methyltransferase-antisense (Bhmt-AS), and examined its expression patterns under pathophysiological conditions. Our results revealed that the expression of Bhmt-AS was significantly increased in the livers of fasted and db/db mice and was induced by gluconeogenic hormonal stimuli. The Bhmt-AS was also shown to be a concordant regulator of Bhmt expression. Functionally, depletion of Bhmt-AS suppressed hepatic glucose production both in vivo and in vitro. Adenovirus-mediated hepatic knockdown of BhmtAS improved pyruvate tolerance, glucose tolerance, and insulin sensitivity. Furthermore, overexpression of Bhmt restored the decreased glucose production caused by knockdown of Bhmt-AS in primary hepatocytes. Taken together, we uncovered a novel antisense lncRNA (Bhmt-AS) that is co-expressed with Bhmt and concordantly and specifically regulates Bhmt expression both in vitro and in vivo to regulate hepatic gluconeogenesis. (C) 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).