Vitamin B12 absorption may be impaired in the setting of hypochlorohydria, and studies have suggested a link between use of proton pump inhibitors (PPIs) and the occurrence of B12 deficiency. We aimed to investigate PPI use as a cause of B12 deficiency. We performed a case controlled retrospective observational study. Medical inpatients aged 65years and older were eligible for inclusion. Vitamin B12 deficient cases (serum B12<150pmol/l) were randomly selected from the preceding year, and age and sex matched non-B12 deficient controls (serum B12>300pmol/l) selected at a ratio of 4:1. Case notes were reviewed for the presence or absence of chronic (>1year) PPI use. Of the 3431 estimations of vitamin B12 status, 168 subjects with B12 deficiency were identified. The case notes of 59 cases and 218 matched controls were reviewed. Twenty-two (35.3%) vitamin B12 deficient cases and 82 (37.6%) controls were taking a PPI. Current use of PPI was not associated with vitamin B12 deficiency (OR 0.92, 95% CI 0.53–1.60). There was no association between PPI dose or duration of therapy and risk of B12 deficiency. Vitamin B12 absorption may be impaired in the setting of hypochlorohydria. However in this older hospital population, vitamin B12 deficiency was not associated with chronic use of PPIs.