BACKGROUND: Vitamin B12 deficiency is highly prevalent among adult individuals with diabetes yet screening is infrequent in Uganda. There are currently no published data regarding the prevalence of vitamin B12 deficiency and its associated factors among adult individuals with diabetes in sub-Saharan Africa. This study aimed at describing the prevalence and factors associated with vitamin B12 deficiency among this patient population in a resource constrained setting in sub-Saharan Africa. METHODS: In this cross-sectional study, 280 eligible study participants attending the outpatient diabetic clinic at Mulago national referral and teaching hospital in Kampala, Uganda were enrolled. Their socio-demographic, clinical and laboratory data was collected using a pre-tested questionnaire. RESULTS: The majority of the study participants were female (68.9 %), with a median age of 50 (IQR: 40-58) years. The mean (SD) serum vitamin B12 levels was 472.0 (16.4) pg/ml. The prevalence of vitamin B12 deficiency was 10.7 %. Hemoglobin level < 12 g/dl (AOR 3.38; 95 % CI 1.38-8.32, p value = 0.008) and glycated hemoglobin ≥ 7 % (AOR 3.29; 1.44-7.51, p value = 0.005) were associated with vitamin B12 deficiency. CONCLUSIONS: Vitamin B12 deficiency is prevalent in approximately 1 in 10 of adult individuals with diabetes in Uganda. We recommend screening for vitamin B12 deficiency among diabetic patients in Uganda especially those with low hemoglobin concentrations and glycated hemoglobin levels ≥ 7 %.