Purpose: This study investigated the influence of moisture on the surface energy of α-lactose monohydrate as determined by inverse gas chromatography. Methods: Dispersive, polar and total surface energies of lactose before and after storage at 75% RH for two months were determined by Inverse Gas Chromatography. The measurements were carried out at infinite dilution with a conditioning RH of 0% for before storage samples and with both 0% and 75% RH for stored samples. The moisture content and the presence of amorphous domains were determined by thermogravimetric analysis and dynamic vapour sorption (DVS), respectively. Results: Compared to before storage, the surface moisture content of α-lactose monohydrate increased by 0.40% w/w after storage at 75% RH for two months. No amorphous content was detected by DVS. The dispersive, polar and total surface energies of the lactose sample before storage were 45.7±1.3 mJ/m2, 150.9±9.0 mJ/m2 and 196.6± 9.9 mJ/m2, respectively. When conditioned for two hours at 75% RH, the dispersive surface energy of the stored sample significantly decreased (P<0.05) while the polar and total surface energies of this sample significantly increased (P<0.05). However, when the surface energy of the stored sample was determined after conditioning for two hours at 0% RH, no significant difference was observed in any of the surface energy parameters. Conclusion: This study concluded that the measured surface energies of stored α-lactose monohydrate samples were different due to the presence of moisture both during storage and conditioning at the storage RH prior to measurement.