The purpose of this study is to analyze the influence of InGaAlP diode laser (660 nm) with or without an odontogenic medium (OM) in the functional activity of OD-21 cells. Undifferentiated OD-21 pulp cells were cultivated with or without OM and divided into four groups (n = 5): nonirradiated control (C -), nonirradiated + OM (C +), irradiated (L -), and irradiated + OM (L +). Laser application was performed in two sessions of a 24-h interval with an irradiance of 11.3 mW/cm2, energy density of 1 J/cm, and total cumulative energy/well of 4.6 J. Cell proliferation, VEGF-164 expression, mineralization, and expression of Alp, Runx2, and Dmp1 genes, as well as immunolocalization of RUNX2 and MEPE proteins, were evaluated. Data were analyzed by statistical tests (α = 0.05). All studied groups showed a similar increase in cell proliferation with or without OM. After 7 and 10 days, a significatively higher concentration of VEGF-164 in L - group when compared to C - group was observed. A significant increase in mineralized nodules in the L + was noted when compared to C + in the same conditions. Photobiomodulation upregulated significantly Runx2 and Dmp1 expression after 10 days in L - and after 7 days in L + , with downregulation of Dmp1 after 10 days in L + group. Immunolocalization of RUNX2 and MEPE was expressive after 7 days of culture in the cytoplasm adjacent to the nucleus with a decrease after 10 days, regardless of the presence of OM. Photobiomodulation enhances metabolism associated with angiogenesis, gene expression, and mineralization regardless of the odontogenic medium in OD-21 cells.