Ticks are obligatory ectoparasites of many vertebrates and transmit pathogens causing diseases such as Heartland virus and Ehrlichiosis. The lone star tick, Ambloyomma americanum L., is the primary vector of Ehrlichia chaffeensis, which causes human monocytic Ehrlichiosis. We aimed to investigate the genomic levels of gene regulation in the processes of acquiring the pathogen and of immune to pathogen. We designed six experimental groups: E. chaffeensis positive and negative groups of males and females, and pathogen free male and female ticks. Illumine HiSeq 2500 sequenced six libraries with 100-cycle single direction. Raw sequence reads (more than 209 million) were trimmed and filtered based on minimum quality score (Q-value >30) and size (> 40nt) for de novo assembly. Assembly using Trinity pipeline produced 140,574 contigs from trimmed and filtered sequence reads (about 117 million reads, 56% of raw data). For quality control of the de novo assembly of transcripts, we filtered out the sequences for mitochondrial, E. chaffeensis, and transposable elements sequences, and tested for contig redundancy and gap separations of the assembled sequences. RSEM and edgeR analyses of 61,802 contigs for identifying differentially expressed genes were followed by Blast2GO analyses for annotations of contigs and enriched-gene ontology (GO) term analyses in pairwise comparisons of the libraries. Further investigation of major groups of genes induced by pathogen would provide better understanding of pathogen-vector interaction, which will allow us to prevent of pathogen transmission by interrupting interaction between pathogen and ticks.