Abstract Background Surfactant therapy has become the standard of care for preterm infants with respiratory distress syndrome. Preclinical studies have reported the therapeutic benefits of mesenchymal stem cells (MSCs) in experimental bronchopulmonary dysplasia. This study investigated the effects of a surfactant on the in vitro viability and in vivo function of human MSCs. Methods The viability, phenotype, and mitochondrial membrane potential (MMP) of MSCs were assessed through flow cytometry. The in vivo function was assessed after intratracheal injection of human MSCs (1 × 105 cells) diluted in 30 μl of normal saline (NS), 10 μl of a surfactant diluted in 20 μl of NS, and 10 μl of a surfactant and MSCs (1 × 105 cells) diluted in 20 μl of NS in newborn rats on postnatal day 5. The pups were reared in room air (RA) or an oxygen-enriched atmosphere (85% O2) from postnatal days 1 to 14; eight study groups were examined: RA + NS, RA + MSCs, RA + surfactant, RA + surfactant + MSCs, O2 + NS, O2 + MSCs, O2 + surfactant, and O2 + surfactant + MSCs. The lungs were excised for histological and cytokine analysis on postnatal day 14. Results Compared with the controls, surfactant-treated MSCs showed significantly reduced viability and MMP after exposure to 1:1 and 1:2 of surfactant:MSCs for 15 and 60 minutes. All human MSC samples exhibited similar percentages of CD markers, regardless of surfactant exposure. The rats reared in hyperoxia and treated with NS exhibited a significantly higher mean linear intercept (MLI) than did those reared in RA and treated with NS, MSCs, surfactant, or surfactant + MSCs. Treatment with MSCs, surfactant, or surfactant + MSCs significantly reduced the hyperoxia-induced increase in MLI. The O2 + surfactant + MSCs group exhibited a significantly higher MLI than did the O2 + MSCs group. Furthermore, treatment with MSCs and MSCs + surfactant significantly reduced the hyperoxia-induced increase in apoptotic cells. Conclusions Combination therapy involving a surfactant and MSCs does not exert additive effects on lung development in hyperoxia-induced lung injury.