"Secondary doping" in poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is quite effective and a frequently used method for the conductivity enhancement. This simple approach, the addition of co-solvents to the PEDOT:PSS solution before film fabrication, is an essential way to derive the high electrical performance of PEDOT:PSS, but the mechanism still remains unclear. In this study, nanoscale structural changes synchronized with the conductivity enhancement via secondary doping in PEDOT:PSS films were investigated. During secondary doping with ethylene glycol near the critical dopant concentration, noncrystalized PEDOT molecules uncoupled from PSS chains and then underwent nanocrystallization. These structural changes might be the key driving force for conductivity enhancement via secondary doping.