Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical technique that can obtain cellular metabolism information based on the intrinsic autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the metabolic state of single cells within the native microenvironment of the living tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in which osteoblastic lineage cells were labelled with green or red fluorescence protein, respectively. Measurement of the mean NAD(P)H lifetime, τ_M, demonstrated that osteoblasts in osteogenic media had a progressively increased τ_M compared to cells in regular media, suggesting that osteoblasts undergoing mineralization had higher NAD⁺/NAD(P)H ratio and may utilize more oxidative phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate cellular metabolism of GFP+ osteoblasts as well as bone tissue oxygen at different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data showed that osteocytes dwelling within lacunae had higher τ_M than osteoblasts at the bone edge of suture and marrow space. Measurement of pO₂ showed poor correlation of pO₂ and τ_M in native bone. However, when NAD(P)H 2P-FLIM was used to examine osteoblast cellular metabolism at the leading edge of the cranial defects during repair in Col (I) 2.3GFP mouse model, a significantly lower τ_M was recorded, which was associated with lower pO₂ at an early stage of healing, indicating an impact of hypoxia on energy metabolism during bone tissue repair. Taken together, our current study demonstrates the feasibility of using non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy metabolism at single cell resolution in living animals. Our data further support that both glycolysis and OxPhos are being used in the osteoblasts, with more mature osteoblasts exhibiting higher ratio of NAD⁺/NAD(P)H, indicating a potential change of energy mode during differentiation. Further experiments utilizing animals with genetic modification of cellular metabolism could enhance our understanding of energy metabolism in various cell types in living bone microenvironment.