<p>Cytochalasans are a class of hybrid polyketide-peptide natural products with diverse activities, including anti-tumor, anti-fungal, anti-parasitic, and anti-HIV properties, demonstrating significant application potential and broad market prospects. With the development of microbial metabolic engineering and synthetic biology, microbial synthesis of cytochalasans has emerged as a cost-effective and efficient alternative to traditional extraction and chemical synthesis methods, facilitating green and sustainable production. To better understand and promote the efficient heterologous biosynthesis of cytochalasans, this review systematically summarizes and discusses the research progress on the biosynthesis of cytochalasans. Firstly, an overview of the classification, application and biological activity of cytochalasans is provided. Subsequently, we systematically review the relevant gene clusters, enzymes, and biosynthetic pathways involved in the biosynthesis of cytochalasans. Additionally, the latest progress in the design of microbial cell factories for producing cytochalasans and the strategies to enhance their performance are summarized and discussed. Finally, the current challenges in developing efficient cell factories for producing cytochalasans with renewable biomass as a substrate and the corresponding strategies are proposed, aiming to achieve higher-efficiency green biomanufacturing of cytochalasans.</p>