A series of vanadium-substituted Cs salt of heteropoly compounds (HPC) from different vanadium-containing precursors were synthesized to investigate the preferable vanadyl species for oxidation of isobutane to methacrylic acid (MAA) under oxygen-poor conditions. The effects of various precursors on the structure were explored by XRD, FT-IR, Raman, UV–vis diffuse reflectance, XPS, H2-TPR, NH3-TPD and IR spectra of pyridine adsorption, and their structure-performance relationship was further studied. It was firstly found that the vanadium-containing precursors played a crucial role in the structure (location of vanadyl species), surface acidity and redox properties (ratio of surface V4+/V5+ and Mo5+/Mo6+ redox couples) for series of as-prepared HPCs catalysts. The catalyst derived from vanadyl sulfate led to vanadyl species into the secondary structure of Keggin-type HPC, which exhibited suitable surface V4+/V5+ and Mo5+/Mo6+ ratio and higher surface acidity, thereby improving the catalytic activity and selectivity to MAA for selective oxidation of isobutane under oxygen-poor conditions. These understandings would be beneficial for further study of HPCs and developing of highly efficient catalysts for isobutane oxidation.