Oncolytic viruses exploited for cancer therapy are developed to selectively infect, replicate, and kill cancer cells to stop tumor growth. However, in some cancer cells, oncolytic viruses are often limited in completing their full replication cycle, making progeny virions, and/or spread in the tumor bed due to the heterogeneous cell types within the tumor bed. Here we report that nuclear export pathway regulates oncolytic myxoma virus (MYXV) infection and cytoplasmic viral replication in a subclass of human cancer cell types where virus replication is restricted. Inhibition of CRM1/XPO-1 nuclear export pathway with nuclear export inhibitors can overcome this restriction by trapping restriction factors in the nucleus and allow significantly enhanced virus replication and killing of human cancer cells. Furthermore, knockdown of CRM1/XPO-1 significantly enhanced MYXV replication in restrictive human cancer cells and reduced the formation of anti-viral granules associated with RNA helicase DHX9. Both in vitro and in vivo, we demonstrate that the approved CRM1 inhibitor drug Selinexor enhances the replication of MYXV and cell killing of diverse human cancer cells. In the xenograft tumor model in NSG mice, combination therapy with Selinexor plus MYXV significantly reduced tumor burden and enhanced the survival of animals. Additionally, we performed global scale proteomic analysis of nuclear and cytosolic proteins in human cancer cells to identify the host and viral proteins that are upregulated or downregulated by different treatments. These results for the first time indicate that Selinexor in combination with oncolytic MYXV can be used as potential new anti-cancer therapy.