Carbohydrate response element binding protein (ChREBP) regulates insulin‐independent de novo lipogenesis. Recently, a novel ChREBPβ isoform was identified. The purpose of the current study was to define the effect of dietary carbohydrates (CHO) and obesity on the transcriptional activity of ChREBP isoforms and their respective target genes. Mice were subjected to fasting‐refeeding of high‐CHO diets. In all three CHO‐refeeding groups, mice failed to induce ChREBPα, yet ChREBPβ increased 10‐ to 20‐fold. High‐fat fed mice increased hepatic ChREBPβ mRNA expression compared to chow‐fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/β activity, HepG2 cells were treated with fructose ± a fructose‐1,6‐bisphosphatase inhibitor to suppress gluconeogenesis. Fructose treatment in the absence of gluconeogenesis resulted in increased ChREBP activity. To confirm the existence of ChREBPβ in human tissue, primary hepatocytes were incubated with high‐glucose and the expression of ChREBPα and ‐β was determined. As with the animal models, glucose induced ChREBPβ expression while ChREBPα was decreased. Taken together, ChREBPβ is more responsive to changes in dietary CHO availability than the ‐α isoform. Diet‐induced obesity increases basal expression of ChREBPβ, which may increase the risk of developing hepatic steatosis, and fructose‐induced activation is independent of gluconeogenesis.