Abstract Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels. Conclusions This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.