Circular RNAs (circRNAs) generated via back-splicing are enhanced by flanking complementary sequences. Expression levels of circRNAs vary under different conditions, suggesting participation of protein factors in their biogenesis. Using genome-wide siRNA screening that targets all human unique genes and an efficient circRNA expression reporter, we identify double-stranded RNA-binding domain containing immune factors NF90/NF110 as key regulators in circRNA biogenesis. NF90/NF110 promote circRNA production in the nucleus by associating with intronic RNA pairs juxtaposing the circRNA-forming exon(s); they also interact with mature circRNAs in the cytoplasm. Upon viral infection, circRNA expression is decreased, in part owing to the nuclear export of NF90/NF110 to the cytoplasm. Meanwhile, NF90/NF110 released from circRNP complexes bind to viral mRNAs as part of their functions in antiviral immune response. Our results therefore implicate a coordinated regulation of circRNA biogenesis and function by NF90/NF110 in viral infection.