Objective To investigate the effect and mechanism of leucine zipper/EF-hand-containing transmembrane protein 1 (LETM1) on proliferation, migration, apoptosis, osteogenic differentiation, and tumorigenesis in vivo of human osteosarcoma MG63 and 143B cells. Methods The osteosarcoma MG63 and 143B cells were divided into blank control group (without adenovirus infection), negative control group (sh-NC group, infected with RNAi negative control virus), and LETM1 knockdown group (sh-LETM1 group, infected with sh-LETM1 adenovirus). Western blotting was performed to detect LETM1 expression in normal human osteoblasts hFOB1.19 and osteosarcoma cells, and to verify the knockdown effect of adenovirus; cell clone formation assays and CCK-8 method were used to detect the proliferation of MG63 and 143B cells; wound-healing assay and Transwell assay were used to test cell migration; DAPI staining and Annexin V-APC/7-AAD flow cytometry double staining were used to detect the apoptosis of MG63 and 143B cells; alkaline phosphatase (ALP) staining and Alizarin Red S staining were used to evaluate early and late osteogenic differentiation of MG63 and 143B cells. Ten nude mice were divided into sh-NC group (n=5, injected subcutaneously into nude mice with 143B cells infected with RNAi negative control virus) and sh-LETM1 group (n=5, injected subcutaneously into nude mice with 143B cells infected with sh-LETM1 adenovirus), and nude mice subcutaneous tumor formation assay was used to examine the in vivo tumor-forming ability of 143B cells in each group. Results Western blotting showed that the expression of LETM1 protein in osteosarcoma MG63 and 143B cells was significantly higher than that in human normal osteoblasts hFOB1.19 (P<0.05), and that the expression of LETM1 protein was markedly reduced after injection with sh-LETM1 adenovirus in MG63 and 143B cells. The results of cell clone formation assay and CCK-8 assay indicated that in MG63 and 143B osteosarcoma cells, the clone formation ability and proliferation ability were significantly reduced in sh-LETM1 group compared with sh-NC group and blank control group (P<0.01). The results of wound-healing assay and Transwell assay demonstrated that in MG63 and 143B osteosarcoma cells, the cell migration rate in sh-LETM1 group was significantly lower than that in sh-NC group and blank control group (P<0.01). DAPI staining and flow cytometry results revealed that the apoptosis rate in sh-LETM1 group was significantly higher than those in MG63 and 143B osteosarcoma cells in sh-NC group (P<0.01). Alkaline phosphatase staining and Alizarin red S staining experiments showed more stained areas and calcium salt nodules in MG63 and 143B osteosarcoma cells in sh-LETM1 group than those in sh-NC group and blank control group. The results of the subcutaneous tumor formation assay in nude mice indicated that subcutaneous tumor formation ability was reduced in 143B sh-LETM1 group compared with 143B sh-NC group. Conclusion LETM1 promotes the proliferation, migration and in vivo tumor formation of MG63 and 143B osteosarcoma cells and the mechanism may be related to the inhibition of apoptosis and osteogenic differentiation.