Cell walls separate individual plant cells. To enable essential intercellular communication, plants have evolved membrane-lined channels, termed plasmodesmata, that interconnect the cytoplasm between neighboring cells. Historically, plasmodesmata were viewed as facilitating traffic of low-molecular weight growth regulators and nutrients critical to growth. Evidence for macromolecular transport via plasmodesmata was solely based on the exploitation of plasmodesmata by plant viruses during infectious spread. Now plasmodesmata are revealed to transport endogenous proteins, including transcription factors important for development. Two general types of proteins, non-targeted and plasmodesmata-targeted, traffic plasmodesmata channels. Size and subcellular location influence non-targeted protein transportability. Superimposed on cargo-specific parameters, plasmodesmata themselves fluctuate in aperture between closed, open, and dilated. Furthermore, plasmodesmata alter their transport capacity temporally during development and spatially in different regions of the plant. Plasmodesmata are exposed as major gatekeepers of signaling molecules that facilitate or regulate developmental programs, maintain physiological status, and respond to pathogens.