Sclerostin antibody (Scl‐Ab) increases bone formation through a process dependent on the activation of canonical Wnt signaling, although the specific signaling in the osteoblast lineage in vivo is largely unknown. To gain insight into the signaling pathways acutely modulated by Scl‐Ab, the transcriptional response of subpopulations of the osteoblast lineage was assessed by TaqMan and microarray analyses of mRNA isolated from laser capture microdissection (LCM)–enriched samples from the vertebrae of ovariectomized rats during the first week after Scl‐Ab administration. Briefly, 6‐month‐old Sprague‐Dawley rats were ovariectomized and, after 2 months, received a single dose of vehicle (VEH) or 100 mg/kg Scl‐Ab (n = 20/group). Lumbar vertebrae were collected at 6, 24, 72, and 168 hours postdose and cryosectioned for LCM. Osteocytes were captured from bone matrix, and osteoblasts and lining cells were captured from bone surfaces based on fluorochrome labeling. mRNA was isolated, amplified, and profiled by TaqMan and microarray. Expression analysis revealed that Scl‐Ab caused strikingly similar transcriptional profiles across all three cell types. Only 13 known canonical Wnt target genes, the majority with known functions in bone, showed a significant change in expression by microarray in response to Scl‐Ab, with Wisp1 and Twist1 being the most responsive. Coincident with increased expression of Wnt target genes was the upregulation of numerous extracellular matrix (ECM) genes. The acute and progressive upregulation of ECM genes in lining cells supports their activation into matrix‐producing osteoblasts, consistent with modeling‐based bone formation. A similar transcriptional profile in osteocytes may indicate that Scl‐Ab stimulates perilacunar/pericanalicular matrix deposition. Pathway analyses indicated that Scl‐Ab regulated a limited number of genes related to cell cycle arrest and B‐cell development. These data describe the acute downstream signaling in response to Scl‐Ab in vivo and demonstrate selected canonical Wnt target gene activation associated with increased bone formation in all mature osteoblast subpopulations. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).