Visceral leishmaniasis (VL), with an estimated incidence of 0.2 to 0.4 million cases worldwide, is the most severe form of Leishmaniasis that may be fatal, if left untreated. In the absence of any vaccine, control of VL relies mainly on chemotherapy. Existing drugs for VL have serious drawbacks in terms of safety, efficacy, cost, administration and development of resistance. Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug efficacy. PMM resistance was induced up to 100µM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed more than 6 fold decrease in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 267 genes (2 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant mutants which included a) reduced oxidative phosphorylation b) increased glycosomal succinate fermentation and substrate level phosphorylation c) reduced DNA synthesis and increased DNA damage repair d) decreased protein synthesis and degradation e) dependency on lipids and amino acids for energy generation f) increased ABC transporters mediated drug efflux. Moreover, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, inhibitors of ABC transporters, MDR1 and ABCG2 respectively. The present study highlights the probable mechanisms of PMM resistance in Leishmania.