A large number of extrachromosomal circular DNAs(eccDNAs) have been found in eukaryotic cells, but the mechanism behind the formation of eccDNAs remains unknown. We previously identified an eccDNA(Chromosome 14: 9,692,083-9,692,623 nt) in the silk gland of Bombyx mori, termed as eccDNA~(fib-L), which can promote the expression of fibroin light chain(fib-L) gene. Here, we explored the precise mechanism underlying the formation of eccDNA~(fib-L). We transfected a DNA fragment containing eccDNA~(fib-L) linear molecule into the cultured cells to construct eccDNA~(fib-L). The molecule was flanked by an upstream sequence of the 5' breaking point of the eccDNA~(fib-L) and a downstream sequence of the 3'breaking point of eccDNA~(fib-L). We found that the eccDNA~(fib-L) was generated in the transfected cells derived from different organisms. The length of the flanking sequences and the direct short repeats "GAGT" flanking the 5' and 3' breaking points of eccDNA~(fib-L) affected the formation efficiency of eccDNA~(fib-L).Moreover, eccDNA~(fib-L) was found to be promoted by apoptosis. Genetics screening and DNA pull-down experiments showed that factors associated with DNA repair, such as DNA polymerase theta(Polθ) for microhomology-mediated end-joining(MMEJ), were involved in the biogenesis of eccDNA~(fib-L).Furthermore, in vitro, a cell-free reaction system containing Polθ was used to validate the key factors involved in eccDNA~(fib-L) iformation. The results showed that Polθ and the direct short repeat "GAGT" were necessary for producing eccDNA~(fib-L). Collectively, our data support a mechanism whereby Polθ can mediate the joining of linear DNA fragments with direct short repetitive sequences at the 5' and 3' ends to form a circular DNA in the silk gland of silkworms. The results not only clarified the essential factors driving eccDNA~(fib-L) formation but also complemented previous reports on eccDNA formation.