The C57BL/6J (B6J) strain is the most widely used mouse strain in metabolic research. B6J mice produce a truncated form of nicotinamide nucleotide transhydrogenase (NNT), an enzyme that pumps protons across the inner mitochondrial membrane. It has been proposed that this results in B6J mice having reduced insulin secretion and glucose intolerance compared with other strains of mice (e.g. C3H/HeH and DBA/2) that have a full-length NNT. The aim of this study was to determine whether truncated NNT was associated with reduced insulin secretion and glucose intolerance, comparing B6 substrains that differ in having a truncated NNT. C57BL/6N (B6N) mice have wild-type Nnt. We compared Nnt expression and activity levels as well as in vivo insulin secretion and glucose tolerance between these mice and B6J. Body weights and specific fat-pad depot masses were alike and Nnt expression and activity levels were similar between B6N and B6J mice. Glucose-mediated insulin secretion and insulin sensitivity were comparable between the two groups of mice, as were plasma glucose and insulin levels during the oral glucose tolerance test. The presence of a truncated Nnt did not affect insulin secretion or glucose tolerance on the C57BL/6 background. We suggest that low or normal levels of NNT (regardless of truncation) have little effect on insulin secretion. Rather, it is the increase in expression of Nnt that regulates and enhances insulin secretion. Our data confirm that B6J is a reasonable control strain for diabetes research; this is especially important considering that it is the strain commonly used to generate genetically modified animals.