Horizontal gene transfer (HGT) plays a key role in bacterial evolution, especially with respect to antibiotic resistance. Fitness costs associated with mobile genetic elements (MGEs) are thought to constrain HGT, but our understanding of these costs remains fragmentary, making it difficult to predict the success of HGT events. Here we use the interaction between P. aeruginosa and a costly plasmid (pNUK73) to investigate the molecular basis of the cost of HGT. Using RNA-Seq, we show that the acquisition of pNUK73 results in a profound alteration of the transcriptional profile of chromosomal genes. Mutations that inactivate two genes encoded on chromosomally integrated MGEs recover these fitness costs and transcriptional changes by decreasing the expression of the pNUK73 replication gene. Our study demonstrates that interactions between MGEs can compromise bacterial fitness via altered gene expression, and we argue that conflicts between mobile elements impose a general constraint on evolution by HGT.