Cold-drawn pearlitic steel wire is widely used because of its high strength and certain toughness. In the process of cold drawing, the evolution of cementite lamellae has an important influence on the strength and toughness of steel wire. In this work, high-carbon steel wire rod with a diameter of 7.81 mm is selected, and the series of high-strength pearlite steel wires with different diameters are obtained by multi-pass cold drawing until the maximum true strain reached to 2.18. The evolution law and microscopic mechanism of cementite lamellae evolution during cold drawing are observed by transmission electron microscope (TEM) and scanning electron microscope (SEM) methods. The results show that during steel wire drawing, the longitudinal fibrous pearlite structure becomes more obvious with the increase of strain, and pearlite lamellae composed of ferrite and cementite are gradually adjusted to the drawing direction, and all of them are parallel to the drawing direction at the strain of 2.18. Complex changes have taken place in the cementite lamellae during drawing, which are characterized by lamellar refinement, bending and fracture in morphology, and polycrystalline, amorphous and nanocrystalline phenomena in microstructure. When the pearlitic lamellar orientation is nearly parallel to the drawing direction, interlamellar spacing gradually decreases, the lamellar arrangement direction gradually turns to the drawing direction, and the cementite lamellae become amorphous and nanocrystalline. When pearlite lamellae are nearly perpendicular to the drawing direction, cementite lamellae are bent, fractured and crystallized. At low drawing strain, dislocations mainly move in ferrite phase with a single slip, forming dislocation lines. At high strain, dislocations transform into multi-slip motions, generating dislocation tangles and dislocation cells, and some dislocations slip across the α-ferrite/cementite interfaces. Dislocation interacts strongly with cementite, and the lattice distortion in cementite increases, which is the main reason for the complex changes of cementite lamellae during cold drawing.