The current study explored the effect of intramuscular injection of vitamin B12 (VB12) in early-lactation dairy cows on subsequent low-moisture part-skim Mozzarella cheese quality and VB12 levels during cheese processing and storage. Twenty-four peripartum dairy cows were blocked based on parity and milk yield and randomly assigned into 2 treatments: basal diet (CON) and basal diet with an intramuscular injection of 10 mg of VB12 per cow per week (VB12). Raw milk was collected to determine VB12 content and then used to make low-moisture part-skim Mozzarella cheese 8 wk after injection. The VB12 content of raw milk and cheese was determined using ultra-performance liquid chromatography coupled with tandem mass spectrometry. We found that VB12 content was significantly increased in milk (15.43 vs. 3.30 ng/mL) and fresh cheese (3.72 ng/g vs. undetectable) from the VB12 group compared with the CON group. However, approximately 70% of VB12 was lost in the whey during cheese making, and no VB12 was detectable in either cheese treatment after 8 wk of storage. Furthermore, no significant differences were observed in fat and protein contents in the cheese between the 2 groups. For cheese color, the b* value increased and the a* value decreased slightly in fresh VB12 cheese. Functional properties of stretchability, flowability, and meltability of VB12 cheese were initially comparable to that of CON cheese, but higher flowability and meltability was observed in VB12 cheese after 8 wk of storage. In summary, intramuscular injection of VB12 in early-lactation dairy cows increases the content of VB12 in milk and fresh cheese with no adverse effect on cheese quality, but substantial VB12 is lost during cheesemaking and declines rapidly during storage. Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.