Stem blister canker, caused by Botryosphaeria dothidea, is becoming the most serious disease of poplar in China. The molecular basis of the poplar in response to stem blister canker is not well understood. To reveal the global transcriptional changes of poplar to infection by B. dothidea, Solexa paired-end sequencing of complementary DNAs (cDNAs) from control (NB) and pathogen-treated samples (WB) was performed, resulting in a total of 339,283 transcripts and 183,881 unigenes. A total of 206,586 transcripts were differentially expressed in response to pathogen stress (false discovery rate ≤0.05 and an absolute value of log2Ratio (NB/WB) ≥1). In enrichment analysis, energy metabolism and redox reaction-related macromolecules were accumulated significantly in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses, indicating components of dynamic defense against the fungus. A total of 852 transcripts (575 upregulated and 277 downregulated transcripts) potentially involved in plant-pathogen interaction were also differentially regulated, including genes encoding proteins linked to signal transduction (putative leucine-rich repeat (LRR) protein kinases and calcium-binding proteins), defense (pathogenesis-related protein 1), and cofactors (jasmonate-ZIM-domain-containing proteins and heat shock proteins). Moreover, transcripts encoding glutathione S-transferase (GST) were accumulated to high levels, revealing key genes and proteins potentially related to pathogen resistance. Poplar RNA sequence data were validated by quantitative real-time PCR (RT-qPCR), which revealed a highly reliability of the transcriptomic profiling data.