The dynamic spheroidization mechanism and its orientation dependence in Ti-6Al-2Mo-2V-1Fe alloys during subtransus hot deformation were studied in this work. For this purpose, hot compression tests were carried out at temperatures of 780–880 °C, with strain rates of 0.001–0.1 s−1. Based on SEM, EBSD and TEM characterization, the results showed that the aspect ratio of the α phase decreased with increasing deformation temperatures and decreasing strain rates. At 880 °C/0.001 s−1, the aspect ratio of the α phase was the smallest at 2.05. The proportion of HAGBs decreased with increasing temperatures and strain rates, which was different from the trend of the spheroidization; this indicated that the formation of HAGBs was not necessary for the spheroidization process. Furthermore, the formation of the α/α interface was related to the evolution of dislocations and twin boundaries at high (880 °C) and low temperatures (780 °C), respectively. Moreover, the dependence of lamellar spheroidization on the crystallographic orientation tilt from the compression direction (θ) was clarified: when θ was between 45° and 60°, both the prism <a> slip and basal <a> slip systems were activated together, which was more favorable for spheroidization. This study could provide guidance for titanium alloy process designs and microstructure regulation.