Sclerostin and parathyroid hormones are strong negative and positive regulators of bone formation, respectively. The anabolic response induced by intermittent (iPTH) treatment is sclerostin status-dependent. However, the interaction between sclerostin and iPTH at the matrix level is unknown. The goal of the current study was to determine if iPTH treatment affects matrix composition and, if so, whether these effects are dependent on sclerostin status. Humeral trabecular and cortical bone sites from 16 week old male wild-type (WT) and sclerostin knockout (KO) mice, which had been treated with vehicle or iPTH from age 10–16 weeks, were examined by micro-computed tomography (µCT) to measure bone volume, backscatter scanning electron microscopy (bSEM) to assess global mineralization, and Fourier transform infrared microspectroscopy (FTIRM) to examine matrix composition (mineral-to-matrix ratio, crystallinity, collagen cross-link ratio, and carbonate substitution). The FTIRM measurements were restricted to the tissue formed during the 6-week treatment period. iPTH treatment led to increased trabecular bone volume (p < 0.001) and this effect was much greater in KO mice than WT mice (interaction effect, p < 0.001). iPTH treatment led to reduced trabecular crystallinity (p = 0.047), increased cortical bone area (p < 0.001), decreased cortical bone crystallinity (p = 0.002) and increased cortical bone collagen cross-linking (p = 0.028) to similar degrees in both WT and KO mice. Compared to WT mice, sclerostin KO mice had higher trabecular and cortical bone mass (p < 0.001) and lower mineral-to-matrix ratio in the trabecular (p = 0.010) and cortical (p = 0.016) compartments. Thus, iPTH-induced changes in bone mass are dependent upon sclerostin status in the trabecular compartment, but not in the cortical compartment. In contrast, iPTH-induced changes in matrix composition are sclerostin-independent in both trabecular and cortical compartments.