As a member of nucleocytoplasmic large DNA viruses, African swine fever virus (ASFV) has been proposed to include an intriguing nuclear stage for DNA replication. In this report, we revisited this early event by using the ASFV type II strain HN09 as a model organism. Our studies began with dissecting the early stages of the ASFV life cycle via analyses by viral growth kinetics, electron microscopy, and quantitative PCR (qPCR). We showed that the single replication cycle lasted about 9 h, with the initial early mRNA transcription taking place around 0.5 h upon virus incubation with host cells, a timing that is much earlier than previously found; the viral DNA replication occurred around 4-5 hours post-infection (hpi), coinciding with the formation of early viral factory (VF), followed by virion morphogenesis at 6-9 hpi. The time-course tracking of viral genomic and newly synthesized DNA via EdU (5-ethynyl-2-deoxyuridine) labeling and DNAscope hybridization combined with 3D reconstruction revealed that the viral DNA remained all the time in the cytoplasm. Further transcriptional studies by RNAscope targeting three early mRNAs (, , and ) and EU labeling of nascent viral RNAs revealed similar results regarding their localization. In line with these observations, disruption of the nucleocytoplasmic shuttling with either small interfering RNA (siRNA) or chemical inhibitor did not affect ASFV replication. In contrast, the replication of porcine pseudorabies virus (PRV) was significantly reduced. Thus, we provide strong evidence to suggest that ASFV DNA replication does not involve a nuclear stage. Our findings provide great insight into the replication biology of ASFV.IMPORTANCEAfrican swine fever virus (ASFV) represents a devastating threat to the global swine industry. This virus has a large genomic size of 170 to 200 kb with a complex virion structure, but how this virus coordinates transcription/replication cascades has remained poorly defined. By using modern techniques, including EdU (5-ethynyl-2-deoxyuridine) and EU labeling, DNAscope and RNAscope, 3D reconstruction, and RNA interference (RNAi), we provide compelling evidence to show that the ASFV life cycle does not involve a nuclear stage, with both viral transcription and DNA replication confined to the cell cytoplasm. Our findings provide important insight into ASFV replication biology and into seeking targets for antiviral drug development.