Simple Summary The sharp decline of laying performance and eggshell quality is a common problem in late period laying hens. Vitamin D-3 (VD3) is a necessary micronutrient which plays an important role in mineral and skeletal homeostasis. With the rapid development of genetic selection, commercial laying hens have increased requirements for performance and nutrients. The commercial supplementary dose of VD3 (62.5 mu g/kg) for late period laying hens may be not enough to satisfy the production. In addition, 25-hydroxyvitamin D-3 (25-OHD3) as an active metabolite of VD3, is a viable alternative to replace VD3. Therefore, the objective of this study was to compare the effects of different supplementary doses and sources between VD3 and 25-OHD3 on the laying performance and eggshell quality in late period laying hens. The results showed that supplementary 125 mu g/kg doses of VD3 or 25-OHD3 had better effects in late period laying hens compared with 62.5 mu g/kg doses of VD3. Additionally, there were no different effects on laying performance or eggshell quality in the hens fed dietary 125 mu g/kg doses of VD3 or 25-OHD3. The objective of this study was to compare high supplementary doses (125 mu g/kg) of vitamin D-3 (VD3) or 25-hydroxyvitamin D-3 (25-OHD3) with commercial supplementary doses (62.5 mu g/kg) of VD3 on laying performance, eggshell quality and ultrastructure, and plasma calcium levels in late period laying hens. A total of 1512 Roman Gray (60-week-old) laying hens were allotted into three treatments with 12 replicates and 42 birds in each replicate. During the 12-week trial period, the layers were fed a basal diet supplemented with different doses of VD3 or 25-OHD3 (62.5 mu g/kg VD3 in control group, CON; 125 mu g/kg VD3 in high level VD3 group, VD3; 125 mu g/kg 25-OHD3 in high level 25-OHD3 group, 25-OHD3). The results showed that high supplementary doses of VD3 or 25-OHD3 increased laying rate (p < 0.05). Moreover, the layers fed high doses of VD3 or 25-OHD3 diets had decreased unqualified egg rate and mortality (p < 0.05). High supplementary doses of VD3 or 25-OHD3 increased eggshell strength and eggshell thickness (p < 0.05). From observation in eggshell ultrastructure, high doses of VD3 or 25-OHD3 diets increased the palisade layer thickness and mammillary knob density (p < 0.05). Furthermore, high doses of VD3 or 25-OHD3 diets increased the calcium levels in plasma (p < 0.05). In summary, compared with 62.5 mu g/kg doses of VD3, supplementary 125 mu g/kg doses of VD3 or 25-OHD3 improved the laying performance, eggshell quality, and plasma calcium levels in late period laying hens. Additionally, there was an equal effect on laying performance and eggshell quality in the hens fed dietary 125 mu g/kg doses of VD3 or 25-OHD3.