Single-stranded RNA bacteriophages (ssRNA phages) infect their hosts by binding to the host receptor pili. Purification of pili usually involves mechanical shearing of pili from cells followed by precipitation. However, previous methods often result in low efficiency or unstable results due to pili retraction. This protocol presents an optimized method for purifying receptor type IV pili from Acinetobacter genomospecies 16 (A. gp16), incorporating enhancements in shearing and collection steps to achieve high yields. We found that repeated passage through syringe needles increases yield, and temperature control is crucial during purification. Additionally, the CsCl density gradient was optimized specifically for this specific strain. The purified type IV pili are suitable for cryogenic electron microscopy (cryo-EM) and various biochemical experiments.