The hole-transporting materials (HTMs) play an important role in energy conversion process of perovskite solar cells (PSCs). Here, two spiro-OMeTAD derivatives, 2,4-spiro-OMeTAD and 3,4-spiro-OMeTAD with asymmetric methoxy substituents, have been designed, synthesized and fully characterized. Optimization of the positions of the two methoxy substituents in each of the quadrants was proved to be able to adjust the electronic and optical properties of the HTMs, as investigated by cyclic voltammetry and UV/vis spectroscopy. In particular, PSC devices based on 2,4-spiro-OMeTAD exhibited highly improved photovoltaic performance showing an overall conversion efficiency of 17.2% with excellent stability, which is higher than that of the device derived from spiro-OMeTAD (15.0%) under the same conditions. Besides, 3,4-spiro-OMeTAD based PSCs only achieved a power conversion efficiency of 9.1%, demonstrating a huge influence of HTM structure on the cell performance.