Duloxetine is a serotonin/noradrenaline reuptake inhibitor that is used as an antidepressant. However, it is known to cause constipation as a side effect. Magnesium compounds, such as magnesium oxide and magnesium hydroxide aqueous solution, are often combined with duloxetine to ameliorate the constipation caused by duloxetine. However, there is concern that these magnesium compounds might alter the effects of duloxetine via physicochemical interactions. In this study, we attempted to clarify the interactions that take place between duloxetine and magnesium oxide using in vivo and in vitro experiments. We evaluated the influence of magnesium oxide on in vitro duloxetine concentrations using HPLC. In addition, we examined the in vivo antidepressant-like effects and serum concentrations of duloxetine in rats. In the in vitro experiment, the duloxetine concentration was significantly decreased by co-treatment with magnesium oxide. In the in vivo experiment, the antidepressant-like effects of duloxetine were not affected by the combined oral administration of magnesium oxide and a duloxetine formulation although the serum duloxetine level was significantly decreased. However, the antidepressant-like effects of a duloxetine reagent were significantly attenuated by the co-administration of magnesium oxide. These results suggest that duloxetine and magnesium oxide directly interact and that such interactions affect the absorption and antidepressant-like effects of duloxetine.