The Golgi apparatus is a highly dynamic organelle which frequently undergoes morphological changes in certain normal physiological processes or in response to stress. The mechanisms are largely not known. We have found that heat shock of Panc1 cells expressing core 2 N-acetylglucosaminyltransferase-M (Panc1-C2GnT-M) induces Golgi disorganization by increasing non-muscle myosin IIA (NMIIA)-C2GnT-M complexes and polyubiquitination and proteasomal degradation of C2GnT-M. These effects are prevented by inhibition or knockdown of NMIIA. Also, the speed of Golgi fragmentation induced by heat shock is found to be positively correlated with the levels of C2GnT-M in the Golgi. The results are reproduced in LNCaP cells expressing high levels of two endogenous glycosyltransferases-core 2 N-acetylglucosaminyltransferase-L:1 and β-galactoside:α2-3 sialyltransferase 1. Further, during recovery after heat shock, Golgi reassembly as monitored by a Golgi matrix protein giantin precedes the return of C2GnT-M to the Golgi. The results are consistent with the roles of giantin as a building block of the Golgi architecture and a docking site for transport vesicles carrying glycosyltransferases. In addition, inhibition/depletion of HSP70 or HSP90 in Panc1-C2GnT-M cells also causes an increase of NMIIA-C2GnT-M complexes and NMIIA-mediated Golgi fragmentation but results in accumulation or degradation of C2GnT-M, respectively. These results can be explained by the known functions of these two HSP: participation of HSP90 in protein folding and HSP70 in protein folding and degradation. We conclude that NMIIA is the master regulator of Golgi fragmentation induced by heat shock or inhibition/depletion of HSP70/90.