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Mesenchymal stem cells (MSCs) are capable of regenerating various mesenchymal tissues and are essential in supporting the growth and differentiation of hematopoietic stem cells within the bone marrow microenvironment in vivo. To achieve clinically meaningful numbers of cells, many approaches have been used to maintain the differentiation potentialities and expand enough cells for clinical treatments. Previously, we have reported that low level light irradiation (LLLI) using 630 nm light emitting diodes (LEDs) could enhance replicative and colony formation potentials of MSCs derived from human bone marrow. The purpose was to study the effect on the proliferation of MSCs derived from the rat bone marrow by red light LLLI (630 nm) under different parameters of irradiation. The irradiance used was 5, 10 or 15 mW/cm2, and the radiant exposure was 2 or 4 J/cm2. Rat MSCs were irradiated at room temperature with single and multiple exposures. The results showed that the proliferation of MSCs plated at the low density (100 cells/well) and high density (1000 cells/well) was enhanced by multiple exposures of red-light LED treatment. The rate of proliferation of MSCs plated at the high density was not as high as those plated at the low density. The optimal parameter for LLLI was at irradiance of 15 mW/cm2, and radiant exposure of 4 J/cm2. The effect on the proliferation of cells by single dose irradiation was temporary. Multiple stimuli may be necessary for the enhancement of cell growth.