Exposure to spaceflight and head-down tilt (HDT) bed rest leads to decreases in the mass of the gluteal muscle. Preliminary results have suggested that interventions, such as artificial gravity (AG), can partially mitigate some of the physiological adaptations induced by HDT bed rest. However, its effect on the gluteal muscles is currently unknown. This study investigated the effects of daily AG on the gluteal muscles during 60-day HDT bed rest. Twenty-four healthy individuals participated in the study: eight received 30 min of continuous AG; eight received 6 × 5 min of AG, interspersed with rest periods; eight belonged to a control group. T1-weighted Dixon magnetic resonance imaging of the hip region was conducted at baseline and day 59 of HDT bed rest to establish changes in volumes and intramuscular lipid concentration (ILC). Results showed that, across groups, muscle volumes decreased by 9.2% for gluteus maximus (GMAX), 8.0% for gluteus medius (GMED), and 10.5% for gluteus minimus after 59-day HDT bed rest (all p < 0.005). The ILC increased by 1.3% for GMAX and 0.5% for GMED (both p < 0.05). Neither of the AG protocols mitigated deconditioning of the gluteal muscles. Whereas all gluteal muscles atrophied, the ratio of lipids to intramuscular water increased only in GMAX and GMED muscles. These changes could impair the function of the hip joint and increased the risk of falls. The deconditioning of the gluteal muscles in space may negatively impact the hip joint stability of astronauts when reexpose to terrestrial gravity.