This study was conducted to examine the effect of dietary rye inclusion and xylanase supplementation on the bone quality of ISA Brown laying hens. Ninety-six laying hens were assigned to four groups: fed with wheat–corn diet or rye–wheat–corn diet (25% of hybrid rye inclusion) or nonsupplemented or supplemented with xylanase (200 mg/kg of feed) for a period of 25 weeks, from the 26th to the 50th week of age. X-ray absorptiometry, X-ray diffraction, and Fourier-transform infrared spectroscopy were used to provide comprehensive information about the structural organization of bone constitutive phases of the tibia mid-diaphysis in hens from all treatment groups. Bone hydroxyapatite size was not affected by diet. Xylanase supplementation influenced the carbonate-to-phosphate ratio and crystallinity index in hens fed with both diets. Xylanase had more pronounced effects on bone mineral density and collagen maturity in hens fed with the rye–wheat–corn diet versus those fed with the wheat–corn diet. The results of this study showed that modern rye varieties, when supplemented with exogenous xylanase, can be introduced to the diet of laying hens without any adverse effects on bone structure.