This study investigates whether regular physical activity (moderate endurance or resistance training twice a week for 3 months) influences the key regulatory molecules of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α), nuclear respiratory factor-1 (NRF1), and mitochondrial transcription factor A (TFAM)) in patients suffering from non-insulin-dependent type 2 diabetes mellitus (T2DM) (n = 16, years = 62 ± 7, body mass index (BMI) = 30 ± 4 kg/m²). Seven T2DM men took part in endurance training, and 9 men participated in resistance training. BMI-matched non-diabetic male control subjects (CON) (n = 7, years = 53 ± 6, BMI = 30 ± 4 kg/m²) were studied for comparison. The protein contents of PGC1α, NRF1, and TFAM were determined using immunohistochemical staining methods on biopsies taken from the musculus vastus lateralis. At baseline, no differences were observed in NRF1-density between the T2DM men and the CON, while the contents of PGC1α and TFAM were decreased in the T2DM men. PGC1α and TFAM contents were not changed in the T2DM patients after the training period, but NRF1 was decreased. The down-regulation of mitochondrial signaling molecules might explain the patho-physiological reduction in mitochondrial biogenesis found in T2DM. Physical training, as performed in our study, did not reverse the down-regulation of mitochondrial signaling molecules — at least not at 3 months after the training.