Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The activity of several enzymes involved in PG synthesis is regulated by serine/threonine kinase (STKs). In firmicutes, inactivation of genes encoding STKs is associated with a range of phenotypes including cell division defects and changes in cell wall metabolism, but only a few kinase substrates have been identified. We previously demonstrated that the STK PrkC plays an important role in cell division, cell wall metabolism and drug resistance in Clostridioides difficile. In this work, we identified and characterized a PGH, CD1135 that is a PrkC substrate. We showed that CD1135 hydrolyses PG between daughter cells to allow cell separation. We demonstrated that CD1135 phosphorylation inhibits CD1135 its export, therefore controlling the hydrolytic activity in the cell wall. High level of CD1135 in the cell wall leads to cell elongation, whereas low level causes cell separation defects. We provided evidences that the STK signaling pathway regulates PGHs homeostasis to control PG hydrolysis during growth and cell division.