Vibrio parahaemolyticus is strongly resistant to ampicillin (AMP). In this study, AMP-resistant genes in V. parahaemolyticus ATCC33846 were characterized. Transcriptomic analysis of V. parahaemolyticus exposed to AMP revealed 4608 differentially transcribed genes, including 670 significantly up-regulated genes and 655 significantly down-regulated genes. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, significantly modulated genes in ATCC33846 under AMP stimulation were observed in the following categories: microbial metabolism in diverse environments, metabolic pathways, bacterial secretion system, citrate cycle, biofilm formation, oxidative phosphorylation, ribosome, citrate cycle, pyruvate metabolism, carbon metabolism, nitrogen metabolism, fatty acid metabolism and tryptophan metabolism. The genes VPA0510, VPA0252, VPA0699, VPA0768, VPA0320, VP0636, VPA1096, VPA0947 and VP1775 were significantly up-regulated at the similar level to blaA in V. parahaemolyticus under AMP stimulation, and their overexpression in V. parahaemolyticus could increase its resistance to AMP. These results indicate that AMP has a global influence on V. parahaemolyticus cells. The findings would provide new insights into the resistant mechanism of V. parahaemolyticus to AMP, which would be helpful for developing novel drugs for treating V. parahaemolyticus infection.