Alms1 mutant (foz/foz) mice develop obesity. Their metabolic phenotype varies depending on genetic background. Female NOD.B10 foz/foz mice fed a high fat (HF) diet for 24 wks exhibit profound hyperinsulinemia with a markedly elevated pro-insulin/c-peptide ratio suggestive of islet β-cell dysfunction, hyperglycemia, an impaired capacity for adipose expansion and non-alcoholic steatohepatitis (NASH). Conversely, HF-fed Balb/c foz/foz mice develop identical obesity, but remain normoglycemic with normal adipose expansion and do not develop NASH. We hypothesized that β-cell dysfunction is an early defect in the HF-fed NOD.B10 foz/foz mice. In this study, we assessed over time insulin secretion in vivo and in vitro in response to HF diet in NOD.B10 foz/foz mice to identify the relationship between initial β-cell dysfunction, hyperglycemia, failure of adipose expansion and onset of NASH. Body, adipose depot and liver weights, fed glucose, insulin, proinsulin and c-peptide, intraperitoneal (ip) glucose tolerance with insulin responses and insulin secretion in isolated islets were measured in chow and HF-fed NOD.B10 and Balb/c wild type (WT) and foz/foz mice after 1-2 wks and 7-8 wks on diet from 4 wks of age. As early as 1 wk on HF diet, NOD.B10 foz/foz mice were glucose intolerant, but without evidence of altered insulin secretion in vivo or in vitro or altered insulin processing. After 7-8 wks of HF diet, NOD.B10 foz/foz mice were diabetic with profound hyperinsulinemia but with an unaltered proinsulin/c-peptide ratio compared to Balb/c foz/foz mice. Furthermore, adipose tissue restriction was not evident at this stage. In conclusion, glucose intolerance is a very early defect in HF-fed NOD.B10 foz/foz mice without evidence of obvious dysfunctional insulin secretion. The mechanism causing this glucose intolerance is unclear but may explain the detrimental metabolic phenotype that develops with diet stressing of NOD.B10 foz/foz obese mice.