Infection with parvovirus B19 (B19) may induce apoptosis, an effect attributed to proapoptotic activity of the non-structural viral protein NS1 which is known to trigger a signaling cascade eventually leading to activation of caspases. Apoptosis was found to be paralleled by profound cytosolic acidification, which may be secondary to inhibition of the Na⁺/H⁺ exchanger. The present study thus explored whether NS1 expression affects cytosolic pH (pHi) and Na⁺-dependent realkalinization (DpHi) following acidification by an ammonium pulse. According to FACS analysis, overexpression of NS1 in B19-inducible cell lines (RXR-10SW) led to activation of caspase 3 and DNA fragmentation. NS1 overexpression resulted in a significant decline of pHi and DpHi, effects significantly blunted by inhibition of caspase 3 with zVAD. Western blotting revealed degradation of NHE1 following NS1 expression. In vitro, caspase 3, but not caspases 6, 7, and 8 degraded NHE1 protein of cell lysates. In conclusion, overexpression of NS1 triggers a signaling cascade eventually leading to activation of caspase 3 and subsequent degradation of NHE1. The effect contributes to cytosolic acidification which may in turn favour activation of caspases and endonucleases and thus may participate in the pathophysiology of B19-infection.