Milled polyacrylonitrile (PAN)-based Carbon Fibers (mPCFs) were prepared from PAN-based carbon fibers by using a ball milling process. The resulting structural changes in the mPCFs were analyzed by correlating the analytical results obtained by X-ray diffraction (XRD) and Raman spectroscopy and verified by transmission electron microscopy (TEM) lattice images and diffraction patterns. The crystallite size La calculated from the XRD measurements decreased as the milling time was increased to 12 h and then decreased as the milling time was further increased to 18 h. The La of both partially-milled Carbon Fiber (pmCF) and milled Carbon Fiber (mCF) calculated from the Raman spectroscopy data continuously increased as the milling time increased. The difference may be because XRD measured the entire sample regardless of pmCF and mCF, while Raman spectroscopy was limited to measuring the surface and differentiated pmCF and mCF. As the ball milling time increased, the fiber surface was firstly broken by the impact energy of the balls, decreasing crystallinity, while the La inside the unbroken fibers increased.