There have been controversial reports on photobiomodulation on cartilage healing so that the research on the potential effect of low intensity He-Ne laser irradiation (HNI) on the proliferation and variation of rabbit cartilage cells in vitro was systematically done in this paper. The chondrocytes isolated from the cartilage sample of 3-week-old New Zealand white rabbits were cultured with newborn calf serum (NCS) at 0%, 2.5%, 5%, and 10%, irradiated by HNI at 5.74 mW/cm2 for 2, 8, 16, 30 and 45 min per day for 6 days, and then incubated till the 13th day. The proliferation and collagen synthesis were assessed by XTT assay and hydroxyproline (Hrp) content measurement, respectively. The DNA expression was observed by acridine orange stain and confocal laser scanning microscope. The chondrocyte morphology and ultrastructure have been observed on the 9th and 13th day after the first HNI by scan electron microscope. There is no significant photobiomodulation (PBM) on the proliferation of the chondrocytes (PPC) cultured with NCS at 10%. There is PPC cultured with NCS at 2.5% or 5%, and the effects are significant when the cells were irradiated by HNI for 16, 30 and 45 min, respectively, among which the radiation time 30 min corresponds the optimum dose 9.42 J/cm2. Hrp secretion increased steadily with days in NHI group. When the photobiomodulation is significant, the chondrocyte DNA expression was significant, and its morphological characteristics have no significant differences from the one cultured with NCS at 10%. There might be photobiomodulation on the proliferation of the chondrocytes cultured in nutritional deficit condition and irradiated by HNI at limited dosage, which is of clinic importance.