Abstract: Insulin is an essential hormone with key roles in energy homeostasis and body composition. Mice and rats, unlike other mammals, have two insulin genes: the rodent-specific Ins1 gene and the ancestral Ins2 gene. The relationships between insulin gene dosage and obesity has previously been explored in male and female Ins2-/- mice with full or reduced Ins1 dosage, as well as in female Ins1-/- mice with full or partial Ins2 dosage. We report herein unexpected hyper-variability in circulating insulin and physiological responses to high fat feeding in male Ins1-/-:Ins2+/- mice. Two large cohorts of Ins1-/-:Ins2+/- mice and their Ins1-/-:Ins2+/+ littermates were fed chow diet or high fat diet (HFD) from weaning and housed in specific pathogen-free (SPF) conditions. Cohort A and cohort B were studied one year apart. Contrary to female mice from the same litters, inactivating one Ins2 allele on the complete Ins1-null background did not cause a consistent reduction of circulating insulin in male mice. In cohort A, HFD-fed males showed an equivalent degree of insulin hypersecretion and weight gain, regardless of Ins2 dosage. In cohort B, Ins1-/-:Ins2+/- males showed decreased insulin levels and body mass, compared to Ins1-/-:Ins2+/+ littermates. While experimental conditions were held consistent between cohorts, we found that HFD-fed Ins1-/-:Ins2+/- mice with lower insulin levels had increased corticosterone. Collectively, these observations highlight the hyper-variability and range of phenotypic characteristics modulated by Ins2 gene dosage, specifically in male mice.