BACKGROUND: The brow area is an important aesthetic unit. Accurate anatomical understanding of the brow is important for clinical treatments related to this region. Due to the complexity of the brow area and limitations of methods, there are still some controversies regarding description of its musculature. OBJECTIVES: In this article, we aimed to reveal the detailed brow musculature with micro-computed tomography (micro-CT) combined with histological staining. METHODS: Eight fresh cadavers (16 hemifaces) were obtained. Eight hemifaces were processed with phosphotungstic acid contrast staining for micro-CT scanning and reconstruction. The corresponding other 8 hemifaces were prepared with Masson staining. RESULTS: The orbicularis oculi muscle, frontalis muscle (FM), depressor supercilii, and procerus muscle were situated superficially. The FM terminated and connected with its antagonistic muscles at 2 to 5 mm above the brow but the fusion line descended in the lateral third. The corrugator supercilii muscle (CSM) originated from the periosteum, extending laterally and superficially. During its course, it mainly penetrated the FM. The CSM had broad subcutaneous insertion, covering about 77.9% ± 7.5% of the hairy brow. CSM fibers connected to the subcutaneous fiber septa to directly transmit contraction force. The soft tissue on the lateral side of the brow was looser than that on the medial side. CONCLUSIONS: Our study deepens the understanding of the precise anatomy of the brow area and helps guide clinical practice.