Adaptation to hypoxia promotes fracture healing. However, the underlying molecular mechanism remains unknown. Increasing evidence has indicated that long non-coding RNAs (lncRNAs) play crucial roles in several diseases, including fracture healing. In the present study, lncRNA microarray analysis was performed to assess the expression levels of different lncRNAs in MC3T3-E1 cells cultured under hypoxic conditions. A total of 42 lncRNAs exhibited significant differences in their expression, including metastasis associated lung adenocarcinoma transcript 1 (MALAT1), maternally expressed 3, AK046686, AK033442, small nucleolar RNA host gene 2 and distal-less homeobox 1 splice variant 2. Furthermore, overexpression of MALAT1 promoted osteoblast differentiation, alkaline phosphatase (ALP) activity and matrix mineralization of MC3T3-E1 cells, whereas its knockdown diminished hypoxia-induced cell differentiation, ALP activity and matrix mineralization in these cells. Moreover, functional analysis indicated that MALAT1 regulated the mRNA and protein expression levels of CCAAT/enhancer binding protein δ by competitively binding to microRNA-22-3p. Adenoviral-mediated MALAT1 knockdown inhibited fracture healing in a mouse model. Taken together, the results indicated that MALAT1 may serve a role in hypoxia-mediated osteogenesis and bone formation.©The Author(s) 2022. Open Access. This article is licensed under a Creative Commons CC-BY International License.