The functionality of canola oil (CO)-based polyol was controlled by esterification reaction. Canola oil-based diol (COBD) afforded thermoplastic polyurethane with interesting microphase separation. COBD had high affinity to interact with hard segments and formed a new crystalline domain for them. COBD reduced the glassy behavior of membranes for gas separation. Low content of COBD caused a simultaneous increase in CO2 permeability and CO2/CH4-CO2/He permselectivities.