Plants sense microbial signatures via the activation of pattern recognition receptors (PPRs), which triggers a range of cellular defences. One response is the closure of plasmodesmata, which reduces symplastic connectivity and the capacity for direct molecular exchange between host cells. Plasmodesmal flux is regulated by a variety of environmental cues but the downstream signalling pathways are poorly defined, especially how cytoplasmic calcium regulates plasmodesmal closure. Here, we identify that flg22-induced closure of plasmodesmata is mediated by a plasmodesmal-localised, Ca2+-binding protein Calmodulin-like 41 (CML41). CML41 mediates rapid callose deposition at plasmodesmata following flg22 treatment in a calcium-dependent manner, independently of other defence responses triggered by flg22 perception. CML41 transcriptionally responds to flg22 and is essential for full defence against Pseudomonas syringae.