Spectinabilin is a rare nitrophenyl-substituted polyketide metabolite produced by Streptomyces spectabilis and S. orinoci, exhibiting antiviral and antimalarial activities. Although its biosynthetic pathway was recently proposed, heterologous production had not been achieved. Here we report the cloning and heterologous expression of the complete spectinabilin gene cluster from S. spectabilis. A fosmid library of S. spectabilis genomic DNA was screened using degenerate primers and sequencing, leading to the isolation of fosmid 79C, which was integrated into the chromosome of S. lividans 66. LC-MS/MS analysis confirmed that the exconjugant produced spectinabilin, while wild-type S. lividans did not. Sequencing of the 45.3 kb insert revealed 14 open reading frames (ORFs) designated spnA-M, encoding type I polyketide synthases (spnA, spnA', spnB, spnC), proteins involved in pNBA starter unit biosynthesis (spnE, spnF, spnG, spnK), post-PKS modification enzymes (spnH, spnI), a transcriptional activator (spnD), and other functional proteins (spnJ, spnL, spnM). Unexpectedly, this gene cluster is evolutionarily closer to the aureothin biosynthetic gene cluster from S. thioluteus than to the spectinabilin cluster from S. orinoci. Moreover, the two nearly identical spectinabilin gene clusters have distinct regulation mechanisms: spnD encodes an AfsR family transcriptional activator, whereas norD from S. orinoci encodes an ArsR family repressor. This study successfully demonstrates the heterologous production of spectinabilin, characterizes its biosynthetic gene cluster, reveals its evolutionary relationships and unique regulatory features, and lays the foundation for further metabolic engineering to increase spectinabilin production and combinatorial biosynthesis of new analogs with improved biological properties.