Cold-drawn pearlitic steel wires are widely used in numerous engineering fields. Combining X-ray line profile analysis and positron annihilation spectroscopy methods, up to 10−5–10−4 vacancies were found in α-ferrite of a cold-drawn pearlitic steel wire with a true strain of e = 3. The formation of deformation-induced vacancies in α-ferrite of cold-drawn pearlitic steel wire was quantitatively testified.