[Background] Ansamitocins, macrocyclic lactam compounds related to maytansine, are synthesized by Actinosynnema pretiosum ssp. auranticun. Based on the differences of ester side chain moieties in C3 position, ansamitocins contain a series of derivatives. Currently, a novel anticancer drug named Kadcyla (T-DM1), formed from ansamitocin P-3 (AP-3), enters the market. T-DM1 had been approved by FDA as an antibody-drug for treating breast cancer. However, the low yield of ansamitocins limits its commercial application. [Objective] To improve AP-3 yield from A. pretiosum ssp. auranticum ATCC 31565, we applied adaptive evolution and addition of betaine. [Methods] The wild-type A. pretiosum ssp. auranticum was used as starting strain to the adverse stress streptomycin and paromomycin. The evolved mutants were screened with better production of AP-3. After 0.1% betaine was added into the fermentation broth of the evolved mutants, the production of AP-3 was further enhanced. Then, gene transcription levels related to AP-3 biosynthesis were analyzed in evolved mutants and starting strain, to investigate the reasons for enhancement of ansamitocin. [Results] Two evolved strains Str16-4-4 and Par16-2-1 have been obtained. After 7 days fermentation, their AP-3 contents were increased by 33.4% and 31.7%, respectively. Addition of 0.1% betaine ultimately resulted in an increase of 54.6% and 47.4% AP-3 production compared to the wild strain. [Conclusion] Through the adaptive evolution strategy, AP-3 production was improved in A. pretiosum ssp. auranticum. This study provides a new strategy to improve the production of AP-3, and also gives a new example for adaptive evolutionary strategy to promote the production of target products.