Loss of functional islet β-cell mass through cellular death or dedifferentiation is thought to lead to dysglycemia during the progression from obesity to type 2 diabetes. To assess these processes in a mouse model of obesity, we performed measures of circulating cell-free differentially methylated insulin II (Ins2) DNA as a biomarker of β-cell death and aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and forkhead box 01 (Foxo1) immunostaining as markers of β-cell dedifferentiation. Eight-week-old, C57BL/6J mice were fed a low-fat diet (LFD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) and were followed longitudinally for up to 13 wk to measure glycemic control and β-cell mass, death, and dedifferentiation. Compared with LFD controls, β-cell mass increased during the feeding period in HFD animals, and statistically greater β-cell death (unmethylated Ins2) was detectable at 2 and 6 wk after diet initiation. Those times correspond to periods when significant step increases in fasting glucose and glucose intolerance, respectively, were detected. ALDH1A3 and Foxo1 immunostaining of the pancreas revealed evidence of β-cell dedifferentiation by 13 wk when fed an HFD, but not in LFD controls. In conclusion, early episodic β-cell death may be a feature of cellular turnover correlated with changes in glycemia during β-cell mass accrual in obesity, whereas β-cell dedifferentiation may be a feature seen later in established disease.—Tersey, SA, Levasseur, EM, Syed, F., Farb, TB, Orr, KS, Nelson, JB, Shaw, JL, Bokvist, K., Mather, KJ, Mirmira, RG Episodic β-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.