Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. Recently, we reported that low doses of OTA promoted PCV2 replication in vitro and in vivo, but the underlying mechanism needed further investigation. The present studies further confirmed OTA-induced PCV2 replication promotion as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. Our studies also showed that OTA induced autophagy in PK-15 cells, as assessed by the markedly increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, autophagy-related protein 5 (ATG5), and Beclin-1 and the accumulation of green fluorescent protein (GFP)-LC3 dots. OTA induced complete autophagic flux, which was detected by monitoring p62 degradation and LC3-II turnover using immunoblotting. Inhibition of autophagy by 3-methylademine (3-MA) and chloroquine (CQ) significantly attenuated OTA-induced PCV2 replication promotion. The observed phenomenon was further confirmed by the knock-down of ATG5 or Beclin-1 by specific siRNA. Further studies showed that N-acetyl-L-cysteine (NAC), an ROS scavenger could block autophagy induced by OTA, indicating that ROS may be involved in the regulation of OTA-induced autophagy. Furthermore, we observed significant increases in OTA concentrations in lung, spleen, kidney, liver and inguinal lymph nodes (ILN) and bronchial lymph nodes (BLN) of pigs fed 75 and 150 μg/kg OTA compared with controls in vivo. Administration of 75 μg/kg OTA significantly increased PCV2 replication and autophagy in the lung, spleen, kidney and BLN of pigs. Taken together, it could be concluded that OTA-induced autophagy in vitro and in vivo promotes PCV2 replication.