The influence of temperature on the extraction kinetics of Cyclosporin A (CyA) from the mycelia of Tolypocladium inflatum was examined in this study. The extraction of CyA from mycelia was performed in a 2-L stirred, baffled vessel using 30% v/v aqueous methanol. The temperature range used was from 5 to 45°C. A linear relationship was found between the extraction yield of CyA and temperature. As the temperature increased, the yield of CyA increased with a maximum CyA yield of 18.3% obtained at 45°C, which is 21.3% higher than the yield at 25°C. The activation energy for the extraction of CyA from T. inflatum was found to be 36.7 kJ/mol, which indicates that the extraction of CyA from T. inflatum is controlled by both solubilization of CyA and diffusion of CyA through the solid phase of mycelia. The overall mass transfer coefficient, kLaS, was found to increase from 1.02 × 10−3 to 1.34 × 10−2 s−1 as the temperature increased from 5 to 45°C. The effective diffusivity of CyA in the solid matrix of mycelia was found to increase from 1.05 × 10−15 to 1.43 × 10−14 m2/s as the temperature increased from 5 to 45°C. A mathematical diffusion model was developed and was used to fit the experimental kinetic data of CyA extraction and determination of CyA effective diffusivities at different temperatures. This is the first time CyA diffusivities as a function of extraction temperature are reported in the literature. Biotechnol. Bioeng. 2007;96:945–955. © 2006 Wiley Periodicals, Inc.