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3D rotation is one of many fundamental manipulations to cells and imperative in a wide range of applications in single cell analysis involving biology, chemistry, physics and medicine. In this article, we report a dielectrophoresis-based, on-chip manipulation method that can load and rotate a single cell for 3D cell imaging and multiple biophysical property measurements. To achieve this, we trapped a single cell in constriction and subsequently released it to a rotation chamber formed by four sidewall electrodes and one transparent bottom electrode. In the rotation chamber, rotating electric fields were generated by applying appropriate AC signals to the electrodes for driving the single cell to rotate in 3D under control. The rotation spectrum for in-plane rotation was used to extract the cellular dielectric properties based on a spherical single-shell model, and the stacked images of out-of-plane cell rotation were used to reconstruct the 3D cell morphology to determine its geometric parameters. We have tested the capabilities of our method by rotating four representative mammalian cells including HeLa, C3H10, B lymphocyte, and HepaRG. Using our device, we quantified the area-specific membrane capacitance and cytoplasm conductivity for the four cells, and revealed the subtle difference of geometric parameters (i.e., surface area, volume, and roughness) by 3D cell imaging of cancer cells and normal leukocytes. Combining microfluidics, dielectrophoresis, and microscopic imaging techniques, our electrorotation-on-chip (EOC) technique is a versatile method for manipulating single cells under investigation and measuring their multiple biophysical properties.