Callose, found in the cell walls of higher plants such as β-1,3-glucan with β-1,6 branches, is pivotal for both plant development and responses to biotic and abiotic stressors. Plasmodesmata (PD), membranous channels linking the cytoplasm, plasma membrane, and endoplasmic reticulum of adjacent cells, facilitate molecular transport, crucial for developmental and physiological processes. The regulation of both the structural and transport functions of PD is intricate. The accumulation of callose in the PD neck is particularly significant for the regulation of PD permeability. This callose deposition, occurring at a specific site of pathogenic incursion, decelerates the invasion and proliferation of pathogens by reducing the PD pore size. Scholarly investigations over the past two decades have illuminated pathogen-induced callose deposition and the ensuing PD regulation. This gradual understanding reveals the complex regulatory interactions governing defense-related callose accumulation and protein-mediated PD regulation, underscoring its role in plant defense. This review systematically outlines callose accumulation mechanisms and enzymatic regulation in plant defense and discusses PD’s varied participation against viral, fungal, and bacterial infestations. It scrutinizes callose-induced structural changes in PD, highlighting their implications for plant immunity. This review emphasizes dynamic callose calibration in PD constrictions and elucidates the implications and potential challenges of this intricate defense mechanism, integral to the plant’s immune system.