AIM: To evaluate the effect of vitamin B12 deficiency anemia (BDA) on peripapillary retinal nerve fiber layer thickness (RNFLT) using spectral domain optical coherence tomography (SD-OCT), and to determine any correlation arising thereof. METHODS: In this cross-sectional observational study, 99 eyes of 50 BDA patients of age 18-65y were compared with 100 eyes of 50 healthy control subjects. All subjects underwent comprehensive clinical, ophthalmic, and hematological evaluation, followed by peripapillary RNFLT assessment using SD-OCT. RESULTS: The mean total, inferior, nasal, and temporal RNFLT were significantly lower in BDA group as compared to control group (P<0.05). The mean total, inferior and nasal RNFLT correlated significantly (P<0.05) with serum Hb%, B12 and mean corpuscular volume (MCV) level (r=0.310, 0.435, -0.386 for total; r=0.932, 0.481, -0.513 for inferior; r=0.344, 0.254, -0.233 for nasal; respectively), while temporal and superior RNFLT quadrant did not show any correlation with any of the hematological parameters (r=0.144, 0.167, -0.096; r=0.111, 0.070, -0.099; respectively). The mean total RNFLT showed progressive thinning at par with the progression of anemia, except in very severe BDA, where an inverse relationship was documented. CONCLUSION: The mean total, inferior, nasal, and temporal peripapillary RNFLT was significantly thinner in BDA patients. Peripapillary RNFLT thinning seemed to proceed at par with the progression of severity of anemia, except in very sever grade. Early assessment of peripapillary RNFLT may be crucial in BDA patients to prevent potential blinding sequelae. Peripapillary RNFLT thinning in BDA patients should be considered in the differential diagnosis of other non-glaucomatous optic neuropathies, as well.