Previous studies using resting-state functional magnetic resonance imaging (fMRI) have found abnormal spontaneous neural activity in patients with major depressive disorder (MDD). Yet, the frequency-dependent neural activity in MDD is largely unknown. Here, we used resting-state fMRI and regional homogeneity (ReHo) methods to investigate spontaneous neural activity in specific frequency bands of 31 MDD patients and 31 age-, gender- and education-matched healthy controls. We examined spontaneous neural activity in three frequency bands: slow-4 (0.027–0.073 Hz), slow-5 (0.010–0.027 Hz), and the typical band (0.01–0.08 Hz). Compared to controls, MDD patients showed increased ReHo in the middle frontal gyrus (MFG) and decreased ReHo in the fusiform and postcentral gyrus at the typical band. Importantly, MDD patients showed increased ReHo in the middle occipital gyrus (MOG) and decreased ReHo in the anterior cingulate cortex (ACC), inferior frontal gyrus (IFG), superior frontal gyrus (SFG) and the bilateral thalamus in the slow-4 band, while they showed increased ReHo in the medial prefrontal cortex (mPFC) in the slow-5 band. Our results suggest that the abnormality of ReHo in MDD is associated with the frequency band and that future studies should take frequency band effect into account when examining spontaneous neural activity.