Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by the expansion of a CAG triplet in the gene encoding for huntingtin (Htt). The resulting mutant protein (mHtt) with extended polyglutamine (polyQ) sequence at the N terminus leads to neuronal degeneration both in a cell-autonomous and a non-cell-autonomous manner. Recent studies identified mHtt in the extracellular environment and suggested that its spreading contributes to toxicity, but the mechanism of mHtt release from the cell of origin remains unknown. In this study, we performed a comprehensive, unbiased analysis of secretory pathways and identified an unconventional lysosomal pathway as an important mechanism for mHtt secretion in mouse neuroblastoma and striatal cell lines, as well as in primary neurons. mHtt secretion was dependent on synaptotagmin 7, a regulator of lysosomal secretion, and inhibited by chemical ablation of late endosomes/lysosomes, suggesting a lysosomal secretory pattern. mHtt was targeted preferentially to the late endosomes/lysosomes compared with wild-type Htt. Importantly, we found that late endosomal/lysosomal targeting and secretion of mHtt could be inhibited efficiently by the phosphatidylinositol 3-kinase and neutral sphingomyelinase chemical inhibitors, Ly294002 and GW4869, respectively. Together, our data suggest a lysosomal mechanism of mHtt secretion and offer potential strategies for pharmacological modulation of its neuronal secretion.SIGNIFICANCE STATEMENT This is the first study examining the mechanism of mutant huntingtin (mHTT) secretion in an unbiased manner. We found that the protein is secreted via a late endosomal/lysosomal unconventional secretory pathway. Moreover, mHtt secretion can be reduced significantly by phosphatidylinositol 3-kinase and neutral sphingomyelinase inhibitors. Understanding and manipulating the secretion of mHtt is important because of its potentially harmful propagation in the brain.