In plants, communication and molecular exchanges between different cells and tissues are dependent on the apoplastic and symplastic pathways. Symplastic molecular exchanges take place through the plasmodesmata (PDs) that connect the cytoplasm of neighboring cells in highly controlled manners. Callose, β-1,3-glucan polysaccharide, is a PD marker molecule that is deposited in cell walls near the PD neck zone and controls PD permeability. During cell differentiation and plant development and in response to diverse stresses, the PD callose level is highly regulated by two antagonistic enzymes, callose synthase (CalS) or glucan synthase-like (GSL) and β-1,3-glucanase (BG). The diverse modes of regulation by CalS and BG have been uncovered in the past decades through biochemical, molecular, genetic and omics methods. This review highlights the recent findings regarding the function of PD callose and the molecular players involved in callose metabolism, and it provides new insight into the mechanisms maintaining PD callose homeostasis.