Inadequate calcium intake during childhood and adolescence is detrimental to bone metabolism. Here, we postulated that calcium supplement prepared from tuna bone with tuna head oil should benefit for skeletal development than CaCO3. Forty female 4-week-old rats were divided into calcium-replete diet (0.55% w/w,S1, n = 8) and low-calcium groups (0.15% w/w for 2 weeks;L;n= 32). ThenLwere subdivided into 4 groups (8/group), i.e., remained onL,L+ tuna bone (S2),S2+ tuna head oil + 25(OH)D3andS2+ 25(OH)D3. Bone specimens were collected at week 9. We found that 2 weeks on low calcium diet led to low bone mineral density (BMD), reduced mineral content, and impaired mechanical properties in young growing rats. Intestinal fractional calcium absorption also increased, presumably resulting from higher plasma 1,25(OH)2D3(1.712 ± 0.158 inLvs. 1.214 ± 0.105 nM inS1,P< 0.05). Four-week calcium supplementation from tuna bone further increased calcium absorption efficacy, which later returned to the basal level by week 9. Calcium supplementation successfully restored BMD, bone strength and microstructure. However, 25(OH)D3+ tuna head oil + tuna bone showed no additive effect. Voluntary running also effectively prevented bone defects. In conclusion, both tuna bone calcium supplementation and exercise are effective interventions for mitigating calcium-deficient bone loss.