Biotinylated carboxylases play essential roles in macronutrient metabolism. We tested the hypothesis that the abundance of biotinylated carboxylases increases in a temporal pattern during the differentiation of mesenchymal stem cells (MSC) into osteoblasts and adipocytes. Cells were collected during early (~10 d) and late stages (~20 d) of differentiation; non‐differentiating MSCs were used as control. The abundance of biotinylated pyruvate carboxylase (PC) and 3‐methylcrotonyl carboxylase (MCC) β‐chain was greater in both early and late stages of osteoblast differentiation compared with non‐differentiating controls; this was apparently caused by an (up to 180%) increase in the abundance of mRNA coding for PC and MCC during differentiation. In contrast, MSC differentiation into adipocytes was accompanied by a substantial (up to 700%) increase in the expression and biotinylation of acetyl‐CoA carboxylase 2, MCC α‐chain, and PC compared with controls, but the increased levels were limited to the early stages of differentiation. We propose that holocarboxylases play an essential role in cell differentiation and currently seek to demonstrate cause‐and‐effect by using biotin‐defined culture media and synthetic inhibitors of biotinylation events. (UNL ARD Hatch Act, NIH DK063945, DK077816, and DK082476)