Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae, pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h−1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1Δ pyc2Δ icl1Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol.