Context: The structural imaging of brain does not demonstrate any changes in the vast majority of patients with vitamin B12 deficiency, even in the advanced stages. Aims: We investigated the microstructural changes in the brain with diffusion imaging among patients with biochemical evidence of B12 deficiency. Patients and Methods: We retrospectively analyzed all diffusion-weighted MRI images between the periods 2014-2016 who had biochemical evidence of B12. The age-sex matched controls were chosen from the group with normal B12 levels. Patients with pathological findings in conventional MRI images were excluded from the study. Results: About 37 patients were recruited (22 women, 15 men; mean age, 34.1 +/- 9.9 years; age range). They were about thirty-four age-and sex-matched controls (with normal B12 levels), which were also included in the study. The mean apparent diffusion coefficient (ADC) value of amygdala (773.8 +/- 49.9 vs. 742.2 +/- 24.2, P = 0.01), hypothalamus (721.3 +/- 39.2 vs. 700.2 +/- 38.2, P = 0.02), striate cortex (737.6 +/- 77.6 vs. 704.3 +/- 58.2, P = 0.04), suprafrontal gyrus (740.7 +/- 46.9 vs. 711.6 +/- 40.7, P = 0.007) and medulla oblongata-olivary nucleus (787.3 +/- 56.4 vs. 759.7 +/- 46.2, P = 0.02) were significantly higher in B12 deficiency group compared to controls, whereas ADC values were similar at hippocampus, thalamus, insula, corpus striatum, cingulate gyrus, occipital gyrus, dentate nucleus, cerebral pedicle, tegmentum, pons, and posterior medulla oblongata. Conclusions: Our study indicates that a significant increase in ADC values occurs in multiple brain regions in patients with vitamin B12.