BACKGROUND Given the unavailability of reliable biomarkers for vitamin B₁₂ (VB₁₂) deficiency in clinical settings, the usefulness of the ¹³C-propionate breath test (PBT), utilizing VB₁₂ as a coenzyme of methylmalonyl-CoA in propionate metabolism, as a diagnostic modality for VB₁₂ deficiency has been studied. However, a collection time of 2 h reduces its convenience. Hence, we evaluated the effectiveness of 1-h PBT for detecting VB₁₂ deficiency in 49 patients with suspected VB₁₂ deficiency. MATERIAL AND METHODS We collected 100-200 mL breath gas every 10 min until 1 h after the administration of 1 g of ¹³C-propionate from 49 patients (31 men, 18 women; median age, 70 years) with clinically suspected VB₁₂ deficiency and calculated the ¹³CO₂ recovered in the breath per hour as the recovery rate (RR [%dose/h]) from ¹³CO₂/¹²CO₂ using infrared isotope spectrometry. We compared the RRs between groups: (1) with serum VB₁₂ levels ≥145 pg/mL and <145 pg/mL, (2) with mean corpuscular volume ≤100 fL and >100 fL, and 3) pre- and post-VB₁₂ supplementation. RESULTS The RRs peaked within 30 min. The RRs at 20 min (RR20) and 30 min (RR30) were significantly lower in macrocytotic patients (41.28 vs 50.07, p=0.026 and 37.82 vs 43.93, P=0.003). The RR30 was higher in the supplemented patients (41.93 vs 32.84, P=0.024). There was no significant difference in RRs between the patients with normal and low serum VB₁₂ levels. CONCLUSIONS The 1-h PBT can be a diagnostic modality for VB₁₂ deficiency because 1 h is a sufficient collection time.