Lactoferrin (LF) increases the calcification of the extracellular matrix by osteoblasts, suggesting a potential for LF in bone formation. The present study evaluates the bone regenerative effect of LF in non-critical-sized rat calvarial flat bone defects using in vivo microfocus computed tomography (micro-CT) and tissue sections. Ten 11-week-old male Fischer rats were used. After establishment of general anesthesia, local anesthesia was performed. A horseshoe-shaped skin incision over the head was made, the parietal area was exposed under aseptic conditions, and the periosteum was elevated to expose the bone. Non-critical-sized calvarial bone defects (each 2.7 mm in diameter) were trephined into the dorsal part of the parietal bone on each side of the midsagittal suture. An absorbable collagen sponge permeated with 5.5 mg of LF was placed on the experimental side. A collagen sponge permeated with saline was placed on the control side. Skin closure was accomplished. The day of surgery was designated as day 0. Repeated micro-CT imaging was performed from 1 to 4 weeks after surgery. The defect sites were removed along with surrounding bone and soft tissues, which were stained with hematoxylin and eosin. At 3 and 4 weeks, micro-CT images showed a significant difference in the reossification ratio. Histological analysis revealed more osteoblast-like cells around the bony rim on the LF side than on the control side. LF accelerated bone regeneration in non-critical-sized rat calvarial bone defects, as confirmed by micro-CT and histological analysis.