The anticoccidial salinomycin is a polyketide produced by Streptomyces albus, and the high-yield strain BK 3-25 produces 18.0 g/L salinomycin under lab condition. In order to elucidate the overproduction mechanism, the genome of BK 3-25 was fully sequenced and compared with the wild-type DSM 41398. Strain BK 3-25 has a 75-kb large deletion, containing type-I polyketide gene cluster PKS-9, and 60 additional InDels and SNVs affecting 55 CDSs, including a 1-bp deletion in type-I PKS gene cluster PKS-6. Subsequently, individual or combined deletions of the 75-kb region and PKS-6 in the wild-type resulted in improved salinomycin yields from 2.60 to 5.20, 6.90, and 9.50 g/L (53% of BK 3-25), respectively, suggesting a redirected flux of polyketide precursors to salinomycin biosynthesis. Moreover, due to the much higher transcription of salinomycin biosynthetic genes (sln) in the high-yield BK 3-25 than in the wild-type, 13 putative regulatory genes among the 55 CDSs were individually inactivated and 7 were proved to be negatively involved in the transcription of sln genes. Combined deletions of two major negative regulatory genes SLNWT_3357 and SLNWT_7015 caused further improved transcription of sln genes as well as the yield, from 2.60 to 7.30 g/L (40% of BK 3-25). Therefore, the comparative genomics approach combined with functional experiments identified that the multiple deletions and mutations of competing gene clusters and negative regulatory genes are crucial for salinomycin overproduction, setting an example for rational titer improvement of other polyketide natural products.