Membrane-anchored Neuregulin β1 sheds its ectodomain as soluble factors. Two proteases that belong to a disintegrin and metalloprotease (ADAM) family are known to cleave Neuregulin β1. One is tumor necrosis factor-α converting enzyme (TACE/ADAM17). The other is Meltrin β (ADAM19). Against our expectation that shedding by ADAM proteases occurs at the cell surface, here we found that Meltrin β mediates the ectodomain shedding of Neuregulin β1 in the Golgi apparatus. Meltrin β was localized in and around the Golgi apparatus in developing sensory neurons. Subcellular fractionation revealed that Meltrin β generated soluble Neuregulin β1 in Golgi-enriched fractions while TACE-cleaved Neuregulin β1 was recovered in lighter fractions. To examine whether Meltrin β-mediated ectodomain shedding occurs in the Golgi apparatus in living cells, we took advantage of different diffusion properties of cleavage products from those of membrane-anchored precursor proteins. Fluorescence correlation spectroscopy (FCS) is the most sensitive method to determine milli∼submillisecond diffusion in vivo. Protease-active Meltrin β caused a shift in autocorrelation function in FCS of green fluorescent protein (GFP)-tagged Neuregulin β1 in the Golgi apparatus, suggesting a conversion of Neuregulin β1 molecules from membrane-anchored to soluble forms in that organelle. The Golgi apparatus is a site of processing Neuregulin β1 by Meltrin β.