We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is ∼94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg2+ or Mn2+ and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (Km 2.6nM) as compared to human DNA ligase I and kcat (2.3×10−3s−1) and kcat/Km (8.8×105M−1s−1) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA–DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.