The objective of this study was to investigate the effects of pathogenic bacterial challenge after acute sublethal ammonia-N exposure on heat shock protein 70 expression in Botia reevesae. After ammonia-N exposure at a constant concentration of 7.21 ± 0.10 mg L−1 for 96 h, B. reevesae was challenged with Aeromonas hydrophila. Quantitative PCR analysis showed predominant and significant expression of HSP70 in liver, gill, skin, spleen and kidney (P < 0.05), with significantly upregulated expression of the mRNA transcript in these tissues after sublethal ammonia-N exposure and A. hydrophila challenge. Furthermore, following A. hydrophila challenge after ammonia-N exposure, HSP70 mRNA expression was significantly upregulated in kidney and gill tissues, although its expression levels were significantly lower than those detected following A. hydrophila challenge or ammonia-N exposure individually. These results indicate that B. reevesae HSP70 is involved in resistance to pathogenic bacteria. It is hypothesized that ammonia-N results in the downregulation of HSP70 mRNA in immune organs after an A. hydrophila challenge, thus lowering their resistance to pathogenic stress.