Background: Liposomal amphotericin B (AmBisome®) as a treatment modality for visceral leishmaniasis (VL) has had significant impact on patient care in some but not all regions where VL is endemic. As the mode of action of AmBisome® in vivo is poorly understood, we compared the tissue-specific transcriptome in drug-treated vs untreated mice with experimental VL. Methods: BALB/c mice infected with L. donovani were treated with 8mg/kg AmBisome®, resulting in parasite elimination from liver and spleen over a 7-day period. At day 1 and day 7 post treatment (R x+1 and R x+7), transcriptomic profiling was performed on spleen and liver tissue from treated and untreated mice and uninfected mice. BALB/c mice infected with M. bovis BCG (an organism resistant to amphotericin B) were analysed to distinguish between direct effects of AmBisome® and those secondary to parasite death. Results: AmBisome® treatment lead to rapid parasitological clearance. At R x+1, spleen and liver displayed only 46 and 88 differentially expressed (DE) genes (P<0.05; 2-fold change) respectively. In liver, significant enrichment was seen for pathways associated with TNF, fatty acids and sterol biosynthesis. At R x+7, the number of DE genes was increased (spleen, 113; liver 400). In spleen, these included many immune related genes known to be involved in anti-leishmanial immunity. In liver, changes in transcriptome were largely accounted for by loss of granulomas. PCA analysis indicated that treatment only partially restored homeostasis. Analysis of BCG-infected mice treated with AmBisome® revealed a pattern of immune modulation mainly targeting macrophage function. Conclusions: Our data indicate that the tissue response to AmBisome® treatment varies between target organs and that full restoration of homeostasis is not achieved at parasitological cure. The pathways required to restore homeostasis deserve fuller attention, to understand mechanisms associated with treatment failure and relapse and to promote more rapid restoration of immune competence.