Elevated serum levels of trimethylamine N-oxide (TMAO) were first associated with increased risk of cardiovascular disease (CVD) 10 years ago. Research has since defined that serum TMAO accumulation is controlled by the diet-microbiome-liver-kidney axis. Choline related nutrients are consumed in excess during over-nutrition from a Western diet. The resultant elevated serum TMAO is investigated across various chronic metabolic diseases and many tissue types. While TMAO is most clearly linked to CVD mechanisms in vascular tissue, its molecular effects on metabolic tissues are unclear. Here we report the current standing of TMAO research in metabolic disease context across relevant metabolic tissues including liver, kidney, brain, adipose, and muscle tissues. This review explores the variable TMAO effects in healthy and diseased conditions. Since impaired pancreatic β-cell function is a hallmark of metabolic disease pathogenesis which are largely unexplored in TMAO research, the following primary research results investigate TMAO effects on in vitro functional β-cell mass in relation to healthy and type 2 diabetes (T2D) conditions. Although we hypothesized that TMAO would aggravate functional β-cell mass, the data demonstrate that TMAO improves the T2D phenotype by increasing insulin secretion and production and reducing oxidative stress. Therefore, this work provides crucial support for the emerging context dependent molecular effects of TMAO during metabolic disease progression.