Pear has an S‐RNase‐based gametophytic self‐incompatibility (SI) system. Nuclear DNA degradation is a typical feature of incompatible pollen tube death, and is among the many physiological functions of vacuoles. However, the specific changes that occur in vacuoles, as well as the associated regulatory mechanism in pear SI, are currently unclear. Although research in tobacco has shown that decreased activity of diacylglycerol kinase (DGK) results in the morphological change of pollen tube vacuole, whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response, is currently unclear. We found that DGK activity is essential for pear pollen tube growth, and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear. Specifically, incompatible S‐RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V‐ATPase V0 domain a1 subunit gene expression as early as 30 min after treatment, when the pollen tube is still alive. Cytoplasmic acidification induced by incompatible S‐RNase results in reduced DGK4 abundance and deposition, leading to morphological change of the vacuole and fragmentation of nuclear DNA, which indicates that DGK4 is a key factor in pear SI response. In pear self‐incompatibility, cytoplasmic acidification of incompatible pollen tubes due to S‐RNase‐mediated inhibition of V‐ATPase V0 dominant a1 subunit gene expression reduces diacylglycerol kinase 4 (DGK4) abundance and deposition, leading to morphological changes of the vacuole and nuclear DNA fragmentation, indicating that DGK4 is a key factor in pear self‐incompatibility.