The methylotrophic yeast Pichia pastoris is one of the most widely used expression systems for the secretory expression of heterologous proteins. This study describes the characterization of the extracellular glycerol kinase (GK) enzyme from the genetically modified P. pastoris yeast. Growth in buffered methanol-complex BMMY medium showed 0.640 U/mL of extracellular GK activity after 48 h, during exponential growth of the cells. A two-enzymatic reactions system comprising glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) was employed for the determination of glycerol kinase activity. Maximum GK activity was achieved with 2.5 µM of substrate (glycerol) and the enzyme diluted 30 times. The enzyme activity fully persisted up to 50 °C for one hour. At pH 7.0 and 30 ºC, its activity persisted for five days. GK followed the Michaelis-Menten kinetics with a Michaelis-Menten constant K_m of 0.11 µM and estimated V_max of 0.058 U/mg of protein. Response surface methodology was used to model enzymatic activity as a function of reaction temperature and pH. The highest activity values were achieved at higher pH values (10.0-10.5) and lower temperatures (40-48 ºC) or higher temperatures (55-60 ºC) and lower pH values (9.0-9.5), with no maximum within the experimental region considered in the present study.