The Sir2 enzyme from Plasmodium falciparum ( Pf Sir2A) is essential for the antigenic variation of this parasite, and its inhibition is expected to have therapeutic effects for malaria. Selective Pf Sir2A inhibitors are not available yet, partially due to the fact that this enzyme demonstrates extremely weak in vitro deacetylase activity, making the characterization of its inhibitors rather challenging. In the current study, we report the biochemical characterization and inhibitor discovery for this enzyme. Pf Sir2A exhibits greater enzymatic activity in the presence of DNA for both the peptide and histone protein substrates, suggesting that nucleosomes may be the real substrates of this enzyme. Indeed, it demonstrates robust deacetylase activity against nucleosome substrates, stemming primarily from the tight binding interactions with the nucleosome. In addition to DNA/nucleosome, free fatty acids (FFAs) are also identified as endogenous Pf Sir2A regulators. Myristic acid, a biologically relevant FFA, shows differential regulation of the two distinct activities of Pf Sir2A: activates deacetylation, but inhibits defatty-acylation. The structural basis of this differential regulation was further explored. Moreover, synthetic small molecule inhibitors of Pf Sir2A were discovered through the screening of a library of human sirtuin regulators. The mechanism of inhibition of the lead compounds were investigated. Collectively, the mechanistic insights and inhibitors described in this study will facilitate the future development of small molecule Pf Sir2A inhibitors as antimalarial agents.