Bisphosphonates are antiresorptive drugs commonly used to treat osteoporosis. It is not clear, however, what the influence of the time point of treatment is. Recently developed in vivo micro-computed tomographic (CT) scanners offer the possibility to study such effects on bone microstructure in rats. The aim of this study was to determine the influence of early and late zoledronic acid treatment on bone in ovariectomized rats, using in vivo micro-CT. Twenty-nine female Wistar rats were divided into the following groups: ovariectomy (OVX, n = 5), OVX and zoledronic acid (ZOL) at week 0 (n = 8), OVX and ZOL at week 8 (n = 7), and sham (n = 9). CT scans were made of the proximal tibia at weeks 0, 2, 4, 8, 12, and 16; and bone structural parameters were determined in the metaphysis. Two fluorescent labels were administered to calculate dynamic histomorphometric parameters. At week 16, all groups were significantly different from each other in bone volume fraction (BV/TV), connectivity density, and trabecular number (Tb.N), except for the early ZOL and control groups which were not significantly different for any structural parameter. After ZOL treatment at week 8, BV/TV, structure model index, Tb.N, and trabecular thickness significantly improved in the late ZOL group. The OVX and ZOL groups showed, respectively, higher and lower bone formation rates than the control group. Early ZOL treatment inhibited all bone microstructural changes seen after OVX. Late ZOL treatment significantly improved bone microstructure, although the structure did not recover to original levels. Early ZOL treatment resulted in a significantly better microstructure than late treatment. However, late treatment was still significantly better than no treatment.