Metabolic engineering exploits manipulation of catalytic and regulatory elements to improve a specific function of the host cell, often the synthesis of interesting chemicals. Although naturally occurring pathways are significant resources for metabolic engineering, these pathways are frequently inefficient and suffer from a series of inherent drawbacks. Designing artificial pathways in a rational manner provides a promising alternative for chemicals production. However, the entry barrier of designing artificial pathway is relatively high, which requires researchers a comprehensive and deep understanding of physical, chemical and biological principles. On the other hand, the designed artificial pathways frequently suffer from low efficiencies, which impair their further applications in host cells. Here, we illustrate the concept and basic workflow of retrobiosynthesis in designing artificial pathways, as well as the most currently used methods including the knowledge- and computer-based approaches. Then, we discuss how to obtain desired enzymes for novel biochemistries, and how to trim the initially designed artificial pathways for further improving their functionalities. Finally, we summarize the current applications of artificial pathways from feedstocks utilization to various products synthesis, as well as our future perspectives on designing artificial pathways.