Low-level laser therapy (LLLT) has been used in photobiomodulation to promote healing, regenerate, and restore tissue function. Women with persistent thin endometrium were assumed to encounter diminished activity in the regenerative and functional capacity of their endometrium. The aim of this study was to examine the ability of LLLT in 635 nm wavelength to enhance the proliferation and gene expression of in vitro cultured endometrial cells. Single (SE) versus multiple exposures (ME) to LLLT were examined in the study groups and compared to controls. A fluence dose of 4.27 J/cm for 16 min was given once in the SE or divided in three equal sessions in days 2, 4, and 6 of the culture in ME. Cellular response was evaluated by measuring viable cell numbers and surface area. Pattern of MUC1, ITGA5, ITGB3, LIF, and PTEN gene expression was assessed using the qRT-PCR. Greater numbers of cells were found in both study groups (P < 0.001) as compared to controls. The surface area of cells at the end of culture phase was highly significant (P < 0.001) in ME when compared to SE and controls. A statistically significant difference was found in terms of gene expression in both irradiated groups (P < 0.001) as compared to controls, although greater difference in PTEN tumour suppressor gene (Phosphatase and tensin homolog) expression was toward ME. The introduction of LLLT to the armamentarium of infertility will serve as a new adjuvant therapy in this field. The current study proofed that LLLT was able to increase the proliferative and functional capacity of cultured endometrial cells.