Hollow glass microspheres (HGMs) can be used as thermal insulation materials owing to its hollow structure which brings excellent thermal insulation property and low density. At present, most researches on thermal conductivity of HGMs are focused on polymer matrix/HGMs composite materials. However, thermal conductivity of HGMs at vacuum in room temperature has rarely been investigated. In this work, thermal conductivity of six types of HGMs (T17 (0.17g/cm3), T20 (0.20g/cm3), T22 (0.22g/cm3), T25 (0.25g/cm3), T32 (0.32g/cm3) and T40 (0.40g/cm3)) at vacuum in room temperature were calculated by heat transfer of solid conduction and radiation. The calculation results showed that thermal conductivity of HGMs would be decreased by an order of magnitude compared with no vacuum. In order to verify the calculation and study vacuum thermal insulation properties of HGMs, thermal conductivity of above-mentioned HGMs at no vacuum and high vacuum in room temperature were measured by a self-made thermal conductivity measuring apparatus which was based on the transient plane source (TPS) method. The experimental results showed that thermal conductivity of HGMs were in the range of 4.2030E-02 to 6.3300E-02 W/m·K (at no vacuum) and 3.8160E-03 to 4.9660E-03 W/m·K (at high vacuum). The results indicated that experimental thermal conductivity was consistent with the calculation results and both of them were all decreased by 8-13 times at vacuum compared with no vacuum. In addition, the relationship with physical properties and thermal conductivity of HGMs has been discussed in detail. In conclusion, HGMs possess excellent thermal insulation performance at high vacuum in room temperature and have potential to further reduce thermal conductivity at the same conditions.