Abstract: A series of aliphatic anionic waterborne polyurethane nano-dispersions (PUD) with different hard segment content (HSC) were prepared by prepolymer mixing method starting from isophorone diisocyanate (IPDI) and poly(ethylene adipate) glycol. Effects of HSC on the structure and properties of PUD have been studied by FTIR, GPC, DSC and other property tests. The increment in HSC leads to a larger average emulsion particle size, but still below 100 nm，and the size distribution becomes wider. FTIR spectra illustrated that more hydrogen bonded urea groups exist in the polymer structure at higher HSC, and the number average molecular weight has a slightly increase as measured by GPC. According to DSC analyses, the glass transition temperature of hard segment increases while that of soft segment decrease indicating an enhanced phase separation at higher HSC. The tensile strength of resultant film prepared from the PUD has a highest value of 52 MPa at 30% HSC, and the breaking elongation decreases dramatically from 2250% to 864% with the HSC increasing. The water resistance is the best at the highest HSC.