Purpose: To evaluate the effects of caffeine and/or estrogen deficiency on trabecular bone area (TBA) and bone healing in rats. Materials and Methods: Rats were divided into groups (n = 15/group) as follows: control, caffeine, ovariectomy (OVX), and caffeine/OVX. Critical-sized defects were created in the tibiae (57 days after beginning caffeine administration and 43 days after OVX). The intact femurs were evaluated for TBA and the number of positive cells for tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG). In the defects, bone healing, the number of TRAP+ and RANKL/OPG+ cells, and gene expression of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin, and CBP/p300-interacting-transactivator-with-ED-rich-tail-2 (CITED-2) were evaluated. Results: Bone healing was poorer in defects of the caffeine group than in those of the control group. The femurs of the OVX and OVX/caffeine groups presented lower TBAs and higher RANKL/OPG+ cell ratios. The number of TRAP+ cells was higher in femurs of the caffeine group and in defects of the OVX group. The caffeine/ OVX group presented the highest RANKL/OPG+ cell ratio in femurs and defects. The OVX group presented the highest expression of BMP-2, BMP-7, and CITED-2. Conclusion: Caffeine affected bone healing, while estrogen deficiency mainly affected TBA, but no significant deleterious synergic effects of both conditions were observed. Keywords: bone density, bone morphogenetic protein, caffeine, osteoprotegerin, receptor activator of nuclear factor kappa-B ligand (RANKL)