Aims. The aim of our study is to investigate the effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage (MCC) of growing rabbits. Methods. A total of 64 ten-day-old rabbits were randomly divided into two groups according to dietary hardness: normal diet group (pellet) and soft diet group (powder). In each group, the rabbits were further divided into four subgroups by feeding time: two weeks, four weeks, six weeks, and eight weeks. Animals would be injected 5-bromo-2′-deoxyuridine (BrdU) every day for one week before sacrificing. Histomorphometric analysis of MCC thickness was performed through haematoxylin and eosin (HE) staining. Immunochemical analysis was done to test BrdU and Notch-1. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure expression of Notch-1, Jagged-1, and Delta-like 1 (Dll-1). Results. The thickness of MCC in the soft diet group was thinner than the one in normal diet group. Notch-1 was restricted in fibrous layer, proliferative layer, and hypertrophic layer. The expression of Notch-1 increased from two weeks to six weeks and then fell down. Notch-1 in normal diet group was higher than that in soft diet group in anterior part of MCC. The statistical differences of Notch-1 were shown at two, four, and six weeks (p < 0.05). The result of western blot and quantitative real-time PCR (qRT-PCR) showed the expression of Dll-1 and Jagged-1 rose from two to four weeks and started to decrease at four weeks. BrdU distributed in all layers of cartilage and subchondral bone. The number of BrdU-positive cells, which were less in soft diet group, was decreasing along with the experiment period. The significant difference was found at four, six, and eight weeks in anterior and posterior parts (p < 0.05). Conclusion. The structure and proliferation of MCC in rabbits were sensitive to dietary loading changes. The proper mechanical loading was essential for transduction of Notch signalling pathway and development of mandibular condylar cartilage. Cite this article: Bone Joint Res 2021;10(7):437–444