The replication of African swine fever virus DNA is initiated inside the nucleus of host cells, being followed by a longer cytoplasmic replication stage. In face of previous results demonstrating the nucleo-cytoplasmic shuttling activity of ASFV p37 structural protein when considered isolated from the virus infection, we performed a systematic analysis of the subcellular localization of p37 protein in ASFV-infected cells, aiming at identifying the role of the nuclear transport mediated by this protein in the viral replication cycle. We report that the p37 protein of the incoming virions is localized throughout the cell at early times post-infection, concentrated in distinct nuclear regions, while at later times the newly synthesized protein is detected exclusively in the cytoplasm of infected cells. Experiments using leptomycin B and siRNAs targeting the CRM1 receptor demonstrate that the subcellular localization of p37 protein is not affected by inhibition of the CRM1-mediated nuclear export pathway. Finally, results from in situ hybridization experiments show a co-localization of the ASFV DNA and p37 protein in specific nuclear regions at early times post-infection, and in viral factories at later times. Overall, these results support the involvement of p37 protein in the nuclear transport of the viral DNA during ASFV replication cycle.