Humic acid (HA) in well water is associated with Blackfoot disease and various cancers. Previously, we reported that acute humic acid exposure (25-200 μg/mL for 24 hr) induces inflammation in RAW264.7 macrophages. In this study, we observed that prolonged (72 hr) HA exposure (25-200 μg/mL) induces cell-cycle arrest and apoptosis in cultured RAW264.7 cells. We also observed that exposing macrophages to HA arrests cells in the G2/M phase of the cell cycle by reducing cyclin A/B1, Cdc2, and Cdc25C levels. Treating macrophages with HA triggers a sequence of events characteristic of apoptotic cell death including loss of cell viability, morphological changes, internucleosomal DNA fragmentation, sub-G1 accumulation. Molecular markers of apoptosis associated with mitochondrial dysfunction were similarly observed, including cytochrome c release, caspase-3 or caspase-9 activation, and Bcl-2/Bax dysregulation. In addition to the mitochondrial pathway, HA-induced apoptosis may also be mediated through the death receptor and ER stress pathways, as evidence by induction of Fas, caspase-8, caspase-4, and caspase-12 activity. HA also upregulates p53 expression and causes DNA damage as assessed by the comet assay. These findings yield new insight into the mechanisms by which HA exposure may trigger atherosclerosis through modulation of the macrophage-mediated immune system.