Plant protoplasts are derived by controlled enzymatic digestion that removes the plant cell wall without damaging the cell membrane. Protoplasts represent a true single-cell system and are useful for various biochemical and physiological studies. Protoplasts from several agriculturally important crop species can be regenerated into a fertile whole plant, extending the utility of protoplasts from transient expression assays to the generation of stable transformation events. Here we describe procedures for transient and stable transformation of leaf mesophyll protoplasts obtained from axenic shoot cultures of canola (Brassica napus). Key steps including enzymatic digestion for protoplast release, density gradient-based protoplast purification, PEG-mediated transfection, bead-type culturing (sea-plaque agarose and sodium alginate), and the recovery of putative transgenic canola plants are described. This method has been used for double-stranded DNA break-mediated genome editing and for the routine generation of stable transgenic canola events at commercial scale.