The effect of calcination temperature on the catalytic activity of VPO catalyst for one step synthesis acrylic acid with acetic acid and formalin was systematically studied using a fixed-bed tubular micro-reactor. The influence of calcination temperature on physicochemical properties especially crystal phase structure of VPO catalysts was obtained. The catalysts were analyzed and characterized with in situ high temperature XRD (HT-XRD), BET, TEM, TG/DTA, XPS, as well as NH3, CO2-TPD, and pyridine-FTIR methods. Larger special surface area and higher V4+ concentration favored higher conversion and selectivity. The optimum special surface area and IV4+/IV5+ ratio was obtained with controlling calcination temperature. The catalyst deactivation was also explored.