The deactivation and in situ regeneration of anion exchange resin (D261) used as a catalyst in the continuous transesterification of soybean oil with methanol for biodiesel production were studied in a fixed-bed reactor. The chemical and physical structures of the resins were investigated by means of X-ray photoelectron spectrometer (XPS), N-2 adsorption/desorption isotherms, and scanning electron microscope (SEM). The results showed that biodiesel conversion was over 90% within the run time of 4 h but rapidly declined to 23.7% after 8 h. The fouling of organic substances (triglyceride and glycerol) covered on the resin was the main reason leading to the decrease in the biodiesel conversion. The fouling extent on the resin was related to the resin position in the fixed bed. The largest activity loss of the resin occurred at the bottom of the fixed bed. The leakage of OH- groups from the resins, independent of the resin height in the bed, also resulted to the decline of the resin activity. An in situ regeneration method was put forward. The resin regenerated can be restored to the original catalytic activity to perform continuous transesterification for biodiesel production.