The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg2+ and Mn2+ but not Ca2+ and Zn2+. Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.