The aim of this study was to investigate lipofuscin origin in brown adipocytes of hyperinsulinaemic rats and the possible role of lipid peroxidation and iron in this process. Ultrastructural examination revealed hyperinsulinaemia-induced enhancement in the lipofuscin production, accompanied by an increase of mitochondrial damage in brown adipocytes. Extensive fusions of lipid droplets and mitochondria with lysosomes were also observed. Confocal microscopy showed lipofuscin autofluorescence emission in brown adipose tissue (BAT) after excitation at 488 nm and 633 nm, particularly in the insulin-treated groups. The presence and distribution of lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE), in brown adipocytes was assessed by immunohistochemical examination revealing its higher content after treatment with insulin. The iron content was quantified by electron dispersive X-ray analysis (EDX) showing its higher content in the hyperinsulinaemic groups. The ultrastucture of the majority of lipofuscin granules suggests their mitochondrial origin, which was additionally confirmed by their co-localization with ATP synthase. In conclusion, our results suggest that increased lipofuscinogenesis in the brown adipocytes of hyperinsulinaemic rats is a consequence of lipid peroxidation, mitochondrial damage and iron accumulation.