Hot isostatic pressing (HIP) temperature has a significant impact on the service performance of powder metallurgy titanium alloys. In this study, a high-temperature titanium alloy, Ti-6.5Al-3.5Mo-1.5Zr-0.3Si, was prepared under different HIP temperatures (880–1000 °C), and the microstructural evolution and mechanical properties were systematically investigated. The results demonstrated that the HIPed alloys were predominantly composed of more than 80 vol.% α phase and a small amount of β phase, and their phase compositions were basically unaffected by the HIP temperatures. Under the typical single-temperature-maintained HIP (STM-HIP) regime, the microstructure of alloy significantly coarsened as the HIP temperature increased, and the alloy strength exhibited an obvious linear negative correlation with the HIP temperature. On the basis of Hall–Petch relation, the prediction model of grain size was established, and the mathematical equation between HIP temperature and grain size (left( {d = Mleft( {T_{{{text{HIP}}}} - N} right)^{ - 2} } right)) was deduced. Furthermore, a possible evolution mechanism of microstructure was proposed, which could be divided into the decomposition of initial α′ martensite for as-received powder, formation of the globular α grains in prior particle boundaries (PPBs) region, and precipitation of the platelet α grains in non-PPBs region. For these alloys prepared by the dual-temperature-maintained HIP (DTM-HIP) regime, although their tensile properties were comparable to that of alloy prepared by STM-HIP regime with same high-temperature holding stage, higher proportion of globular α grains occurred due to more recrystallization nucleation during the low-temperature holding stage, which probably provided a solution for improving the dynamic service performance of HIPed alloys.