Cell-based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large-volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co-administration of all-trans retinoic acid (ATRA) and human salivary peptide histatin-1 (Hst1) on the spreading and osteogenic activities of pre-osteoblasts on bio-inert glass surfaces. Pre-osteoblasts (MC3T3-E1 cell line) were seeded onto bio-inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point-counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, β, and γ, such as ER-50891, LE-135, and MM-11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short-term (2 h) co-administration of 10 μm ATRA and Hst1 to pre-osteoblasts resulted in significantly higher spreading of pre-osteoblasts compared to ATRA or Hst1 alone. ER-50891 and LE-135 both nullified these effects of ATRA. Co-administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co-administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre-osteoblasts on bio-inert glass surfaces in vitro.