Cortical Layer 1 (L1) acts as a critical relay for processing long-range inputs. GABAergic inhibitory interneurons (INs) in this layer (Layer 1 interneurons [L1INs]) function as inhibitory gates, regulating these inputs and modulating the activity of deeper cortical layers. However, their characteristics and circuits in the medial prefrontal cortex (mPFC) remain poorly understood. Using biocytin labeling, we identified three distinct morphological types of mPFC L1INs: neurogliaform cells (NGCs), elongated NGCs (eNGCs), and single-bouquet cell-like (SBC-like) cells. Whole-cell recordings revealed distinct firing patterns across these subtypes: NGCs and eNGCs predominantly exhibited late-spiking (LS) patterns, and SBC-like cells displayed a higher prevalence of non-LS (NLS) patterns. We observed both electrical and chemical connections among mPFC L1INs. Optogenetic activation of NDNF(+) L1INs demonstrated broad inhibitory effects on deeper layer neurons. The strength of inhibition on pyramidal neurons (PyNs) and INs displayed layer-specific preference. These findings highlight the functional diversity of L1INs in modulating mPFC circuits and suggest their potential role in supporting higher order cognitive functions.