Electro-dewatering (EDW) is considered as one of the most promising dewatering technologies due to saving power consumption. In this study, the potential effects of anaerobic biological conditioning (BC) on sludge EDW treatments was investigated. The results showed that without any additives BC pretreatment of sludge enhanced EDW dewaterability and energy efficiency. At 35 °C BC for 3 days, the dry solids (DS) of sludge dewaterability limit could increase up to 49%, which corresponded to an increase of 13% of DS in dewatered sludge cake without BC pretreatment, and the dewatering time was shortened by 22%. There was an economic advantage saving in energy consumption of around 49.5% in the case of BC-EDW when the DS of sludge was up to 38%. Then, the mechanism of BC to improve EDW performance was studied. The principal component regression (PCR) analysis showed that the DS content of dewaterability limit mainly depended on the degradation of organic matter and the change of conductivity in sludge. Fourier transform infrared spectroscopy (FTIR), zeta potential and bound water in sludge were also determined in an attempt to explain the observed changes in sludge BC-EDW. It was indicated that the increase of negatively charged hydroxyl groups on the surface of sludge particles resulted in an increase of the absolute value of the zeta potential and significantly promoted EDW. The tightly bound EPS (TB-EPS) decreased and it loosened the bond between water or metal cations and sludge particles, and the bound water was also found to be released into free water in sludge during BC.