3-Hydroxypropionic acid (3-HP) is an important intermediate compound in the chemical industry. Green and environmentally friendly microbial synthesis methods are becoming increasingly popular in a range of industries. Compared to other chassis cells,Yarrowia lipolyticapossesses advantages, such as high tolerance to organic acid and a sufficient precursor required to synthesize 3-HP. In this study, gene manipulations, including the overexpression of genesMCR-NCa,MCR-CCa,GAPNSm,ACC1andACSSeL641Pand knocking out bypass genesMLS1andCIT2, leading to the glyoxylate cycle, were performed to construct a recombinant strain. Based on this, the degradation pathway of 3-HP inY. lipolyticawas discovered, and relevant genesMMSDHandHPDHwere knocked out. To our knowledge, this study is the first to produce 3-HP inY. lipolytica. The yield of 3-HP in recombinant strain Po1f-NC-14 in shake flask fermentation reached 1.128 g·L−1, and the yield in fed-batch fermentation reached 16.23 g·L−1. These results are highly competitive compared to other yeast chassis cells. This study creates the foundation for the production of 3-HP inY. lipolyticaand also provides a reference for further research in the future.