The cast solvent effect on the structure and properties of sulfonated poly(ether ether ketone) (sPEEK) was studied. PEEK was sulfonated to have different sulfonation degrees of 65, 70, and 75%, and its membrane was prepared using the two types of solvents, N,N-dimethylacetamide (DMA) and 1-methyl-2-pyrrolidinone (NMP). Ionic cluster size was analyzed using small-angle X-ray scattering (SAXS), and it was correlated with a few essential membrane properties such as water uptake, methanol permeability, proton conductivity, and cell performance in direct methanol fuel cells (DMFCs). Synchrotron SAXS and solid state NMR data revealed the structural difference between the sPEEK membranes prepared using NMP and DMA, regarding the cluster dimensions of 3.22 and 2.70 nm, respectively. Although the water uptake, methanol permeability, and proton conductivity of the membranes prepared with NMP were higher than those with DMA, the overall cell performance was vice versa. The dimensional instability associated with high water swelling as well as high methanol permeability were the main causes for this inferior cell efficiency of NMP cast membranes. This report demonstrates the importance of selection of cast solvent in preparation of SPEEK electrolyte membranes for DMFC application.