There is increasing concern about welfare of laying hens in cages, and one aspect of this topic relates to bone fragility. Therefore, bone anabolic components such as bovine lactoferrin (bLF) may be an effective strategy to maintain the integrity and health of bones. A total of 1080 eggs were divided into four groups with three replicates, each comprising 270 eggs; (1) control group was injected with 100 μl of normal saline per egg; (2, 3 and 4) groups including 22.5 (low), 45 (medium) and 67.5 µg (high) of bLF in 100 µl of normal saline per egg. Eggs were incubated and after hatching, chicks were reared to 28 weeks of age. Tibia measurements were obtained at hatch and at 28 weeks of age. Tibia weight at hatch, was not influenced by in ovo injection of bLF in comparison with the control. Eggs injected with the high concentration of bLF (67.5 µg of bLF per egg) showed significant strengthening in laying-hen tibias at 28 weeks of age, as measured by ultimate force and bending stress, compared with the control. Egg weights from hens treated with this concentration of bLF were also significantly greater than the control. Our data suggest that tibia cortical thickness is a suitable variable for evaluating bone status reflecting bone integrity and strength. The present study also shows that bLF (67.5 µg of bLF per egg) injected into layer breeder eggs before incubation can be used to improve bone strength and egg weight of laying hens at 28 weeks of age, while having no detrimental effect on embryo hatchability.