A series of liquid crystalline polyrotaxanes containing azobenzene mesogenic moieties (AzoPR) with different length of spacer were synthesized, and the relationship between the spacer length and the liquid crystalline behavior was investigated. The molecular characterization of the AzoPR was performed with (1)H NMR, FT-IR, and gel permeation chromatography. The thermal stability was investigated via thermogravimetric analysis. Their phase structures and liquid crystalline properties were studied by differential scanning calorimetry, polarized optical microscopy and wide-angle X-ray diffraction. The experimental results suggested that AzoPR with spacer length of 2 and 4 failed to show the liquid crystalline behavior, and AzoPR with spacer length of 6 showed the columnar nematic phase. However, when the spacer length increases to 11, the columnar nematic phase formed, meanwhile, the liquid crystalline domains with high ordered structure were developed by azobenzene mesogens.