The aim of this study is to investigate the efficiency of using crude glycerol, the main byproduct of biodiesel industry, directly in the production media of Pichia pastoris fermentation processes. Different biodiesel synthesis conditions were examined, that is, transesterifications of canola, corn, soybean, and sunflower oils, with pure methanol in a molar ratio of 1:6 or 1:3, using 1% or 0.5% (w/v) NaOH as the catalyst. Among these, canola oil-derived glycerol served as the most favorable carbon source, and at a 1:6 molar ratio of canola oil: methanol and C-NaOH = 1% (w/v), the highest recombinant human erythropoietin production (C-rEPO = 31 mg L-1), cell concentration (C-x = 10.5 g L-1), product yield on substrate (Y-rEPO/S = 1.48 mg g(-1)), and cell yield on substrate (Y-X/S = 0.57 g g(-1)) were obtained, having values approximately 1.4-fold higher than those attained with pure glycerol. Thus, this study demonstrated a potential use for crude glycerol, without any purification, in the production of additional value-added products.