Pemetrexed is indicated for non-small cell lung carcinoma and mesothelioma, but often has limited efficacy due to drug resistance. To probe the molecular mechanisms underlying chemotherapeutic response, we performed mRNA and microRNA (miRNA) expression profiling of pemetrexed treated and untreated lymphoblastoid cell lines (LCLs) and applied a hierarchical Bayesian method. We identified genetic variation associated with gene expression in human lung tissue for the most significant differentially expressed genes (Benjamini-Hochberg [BH] adjusted p < 0.05) using the Genotype-Tissue Expression data and found evidence for their clinical relevance using integrated molecular profiling and lung adenocarcinoma survival data from The Cancer Genome Atlas project. We identified 39 miRNAs with significant differential expression (BH adjusted p < 0.05) in LCLs. We developed a gene expression based imputation model of drug sensitivity, quantified its prediction performance, and found a significant correlation of the imputed phenotype generated from expression data with survival time in lung adenocarcinoma patients. Differentially expressed genes (MTHFD2 and SUFU) that are putative targets of differentially expressed miRNAs also showed differential perturbation in A549 fusion lung tumor cells with further replication in A549 cells. Our study suggests pemetrexed may be used in combination with agents that target miRNAs to increase its cytotoxicity.