Thyrotrophin-releasing hormone (TRH) and TRH-like peptides have been involved in several central and peripheral functions regulated by a system of interlocked photoperiod-driven feedback loops that could be disrupted by dietary dislocations of modern life such as high cholesterol intake. The functions of these peptides are regulated by pyroglutamyl aminopeptidase (PAP), an omega peptidase widely distributed in fluids and tissues. Here we analyze whether dietary cholesterol modulates the activity of PAP in serum and various tissues of male and female mice, to understand how cholesterol could influence TRH functions through its regulation by PAP enzyme activity, and if gender differences exist in these regulatory mechanisms. Male and female mice were fed with a standard diet or a standard diet enriched with 1% cholesterol and 0.5% cholic acid. PAP activity was measured fluorometrically in serum and in its soluble and membrane-bound forms in liver, heart (atrium and ventricle), lung, kidney, testis, ovary and breast. We found gender differences in PAP activity and in its response to dietary cholesterol in serum and different tissues, which indicates that high cholesterol alters the regulation of this activity on their specific biologically active peptide substrates, mainly TRH and TRH-like peptides, and modifies their specific functions on target organs.