Avian leukosis virus subgroup J (ALV-J) has resulted in considerable economic losses in the poultry industry. In recent years, fibrosarcoma induced by ALV-J, which contains the v-fps oncogene, has gained momentum, and this has brought about new challenges to the poultry industry. To study the inhibitory effects of Taishan Pinus Massoniana pollen polysaccharide (TPPPS) on acute ALV-J infection and tumor development, antiviral and antitumor models of the Fu-J (SDAU1005) strain of ALV-J were established in vitro and in vivo. The results of in vitro experiments showed that TPPPS significantly inhibited viral replication in a dose-dependent manner during adsorption and pretreatment stages. The results of in vivo experiments have shown that TPPPS significantly reduced the viral load in the plasma and tumor tissues, as well as inhibited tumor growth. We further examined the difference in transcriptome expression by using RNA-Seq technology. A total of 560 differentially expressed genes were identified that included 329 up-regulated genes and 231 down-regulated genes. The up-regulated genes were mainly immune-related genes, whereas the down-regulated genes were mainly tumor-regulated genes. Gene Ontology (GO) term enrichment included immune system processes, positive regulation of immune system processes, regulation of immune system processes, leukocyte activation, cell activation, and protein binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the main immune and tumor-related pathways included T-cell receptor signaling pathway, cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity, PI3K-Akt signaling pathway, JAK-STAT signaling pathway, NF-κB signaling pathway, and Ras signaling pathway. In summary, our results preliminarily point to the antiviral and antitumor mechanism of TPPPS in vivo and in vitro.