Newborn mice weaned from mice fed on a B12-deficient diet during pregnancy and lactation were fed on a B12-deficient diet for 90 days after weaning, and the state of B12 deficiency was evaluated. The effect of B12 deficiency on the testicular tissue was also examined. The body weight of the mice fed on a B12-deficient diet for 90 days was slightly lower than that of the control mice administrated CN-B12, and the urinary excretion of methylmalonic acid (MMA) was increased. The B12 concentrations in the liver and testes were markedly depressed by B12 deficiency, being about 13 and 10 pmol/g, respectively, on day 90. The testes weight was clearly reduced by B12 deficiency. The testes weight/100 g body weight was also lowered. Clear morphological changes were observed in the testicular tissue of the B12-deficient mice. These results showed that mice in a severely B12-deficient state could be produced by dietary B12 deprivation. These B12-deficient mice could be useful as model animals not only for elucidating the functions of B12 in vivo, but also for biochemical studies.