Background Human milk vitamin B12 (B12) concentrations depend on maternal status and intake; only few data are available in high-income countries. Objective We assessed human milk B12 concentrations during the first 6 months postpartum in Norwegian women and its association with maternal dietary B12 intake and maternal urinary methylmalonic acid (MMA) concentration. Methods In this cross-sectional study, 175 mothers, exclusively (80%) or partially (20%) breastfeeding, were included. Milk B12 was measured by IMMULITE ® /IMMULITE ® 1000 B12 competitive protein binding assay and urinary MMA relative to creatinine (MMA/Cr) by liquid chromatography-tandem-mass spectrometry. Maternal habitual B12 intake and supplement use were estimated using a food frequency questionnaire. Results Mean human milk B12 concentration was 327 pmol/L (range 140-1089), with 402 pmol/L at 1 month (n = 21), 333 pmol/L at four months (n = 32), and 299 pmol/L at 6 months (n = 21). Maternal B12 intake was 5 µg/d, 89% met the Estimated Average Requirement, and supplement use did not affect milk B12 concentrations. MMA/Cr was low in all women compared with published data. In exclusively breastfeeding women, MMA/Cr (beta (95% CI) −42.5 (−82.5, −2.5) and time since birth (−4.9 (−9.6, −0.3)) were significant predictors of human milk B12 concentrations. There was no association between total B12 intake and milk B12 concentration or between total B12 intake and MMA/Cr. Conclusions Maternal B12 status and human milk B12 concentrations are likely sufficient, based on adequate maternal B12 dietary intake combined with low urinary MMA concentrations. Nevertheless, milk B12 concentration fell during 6 months postpartum while maternal B12 status did not change.