The cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is a key component of the transcription complex regulating glucagon driven hepatic glucose production and previous evidence suggests that “inhibition” of CRTC2 improves glucose homeostasis in multiple rodent models of type 2 diabetes. Here we describe a process of identifying potential therapeutic antisense oligonucleotides (ASOs) directed against CRTC2. These ASOs were designed as locked nucleic acid (LNA) gapmers and a panel of approximately 400 sequences were first screened in vitro within both human and mouse liver cell lines. A group of active and selective compounds were then profiled in acute studies in mice to determine the level of CRTC2 mRNA reduction in liver as well as to obtain a preliminary indication of safety and tolerability. The compounds with the best activity and safety profiles were then evaluated in subchronic efficacy studies using the diet induced obese (DIO) mouse model of type 2 diabetes and primary human hepatocytes. Efficacy findings broadly confirmed the beneficial effect of reducing CRTC2 mRNA levels towards improving glucose control and other markers of metabolic function. Additionally, for the first time, translation to human cells has been established with demonstration of a reduction in glucagon-mediated glucose production in primary human hepatocytes and a potential clinical biomarker source identified to assess modulation of CRTC2 mRNA following ASO treatment. While the compounds identified herein did not demonstrate a therapeutic index sufficient for further development, this study should facilitate more efficient prosecution of compounds within an in vivo setting.