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A series of polycarboxylate-based superplasticizers (PCs) with different structures were synthesized and the effects of chemical structure on zeta potential and rheological property of cement paste were studied. Residual monomers in each sample of PCs were quantitatively determined. The property of the polymers in cement was tested by micro-electrophoresis apparatus and R/S rheometer. Results showed that the zeta potential and its rheological properties are related with the side-chain length and density of PCs. The PCs having shorter side chain and lower side chain density exhibit higher anionic charge density, thus resulting in higher zeta potential. The effect of side chain density on zeta potential is more notable compared with that of side-chain length, and thus affecting the initial shear yield stress and apparent viscosity of the cement paste. In addition, although increasing the side chain length will result in reduction of the anionic charge density, the steric hindrance effect is obvious, which can effectively improve the dispersion of the cement particles, and reduce the viscosity and shear yield stress of slurry.