BackgroundMasquelet’s induced membrane (IM) has osteogenesis activity, but IM spontaneous osteogenesis (SO) has not been described previously.ObjectivesTo report on varying degrees of IMSO and analyze its possible causes.MethodsTwelve eight-week-old male Sprague-Dawley rats with 10 mm right femoral bone defects who received the first stage of IM technique (IMT) were used to observe the SO. In addition, clinical data from patients with bone defects who received the first stage of IMT with an interval of > 2 months post-operatively and exhibited SO between January 2012 and June 2020 were retrospectively analyzed. The SO was divided into four grades according to the amount and characteristics of the new bone formation.ResultsAt twelve weeks, grade II SO was observed in all rats, and more new bone was formed in the IM near the bone end forming an uneven margin. Histology revealed bone and cartilage foci in the new bone. Four of the 98 patients treated with the first stage of IMT exhibited IMSO, including one female and three males with a median age of 40.5 years (range 29–52 years). The bone defects were caused by severe fractures and infection in two cases and by infection or tumor in one case each. Partial or segmental defects occurred in two cases. The time interval between inserting a cement spacer and diagnosis of SO ranged from six months to nine years. Two cases were grade I, and one case each of grades III and IV.ConclusionVarying degrees of SO confirm the existence of the IMSO phenomenon. Bioactive bone tissue or local inflammation and a long time interval are the primary reasons underlying enhancement of the osteogenic activity of IM and leading to SO, which tends to take place as endochondral osteogenesis.