The broadly conserved second messenger nucleotide cyclic di-3’,5’-adenosine monophosphate (c-di-AMP) has been shown to regulate a wide range of bacterial processes. C-di-AMP has been shown to bind to and allosterically regulate the activity of pyruvate carboxylase (PC) in Listeria monocytogenes. The c-di-AMP binding pocket residues are also conserved within PC of the economically important cheese fermentation bacterium Lactococcus lactis. In this work we were interested to determine the role of PC and c-di-AMP inhibition of PC in the physiology of L. lactis. A markerless PC deleted mutant of L. lactis MG1363 background (wt) and c-di-AMP insensitive PC variant overexpression mutants in a high c-di-AMP ΔgdpP strain (OS2) were generated and characterised. The PC deleted mutant exhibited similar growth compared to that of wt in rich media with abundant amino acids, but did not grow in chemically defined media (CDM) in the absence of aspartate or asparagine. Complementation with the wt PC gene into the PC deleted mutant restored growth in CDM. In milk, the PC deleted mutant had a significantly slower acidification rate than that of the complemented strain. Supplementation of aspartate or asparagine to milk completely restored rates of acidification and stationary-phase cell numbers of the PC deleted mutant. In the high c-di-AMP mutant strain OS2, the aspartate level was 65% lower than wt. Overexpression of c-di-AMP insensitive Y715T PC variant restored high aspartate levels comparable to that in the wt. In conclusion PC is essential for aspartate biosynthesis in L. lactis and is required for efficient acidification of milk, a key property of starter cultures. C-di-AMP negatively regulates the aspartate pool in L. lactis, and expression of a c-di-AMP insensitive LlPC can restore normal levels of aspartate.