Protein kinase D1 (PKD1), one of the protein kinase D (PKD) family members, plays a prominent role in multiple bio-behaviors of cancer cells. Low pH and hypoxia are unique characteristics of the tumor microenvironment. The aim of this study was to investigate the role and mechanism of PKD1 in regulating metabolism in the human tongue squamous cell carcinoma (TSCC) cell line SCC25 under a hypoxic condition, as well as growth and apoptosis. Here, we found that hypoxia not only induced the expression of HIF-1α, but also induced the expression and activation of PKD1. Moreover, we inhibited the expression of PKD1 by shRNA interference, and the growth of SCC25 cells under hypoxia was significantly decreased, as well as the expression of HIF-1α, while the percentage of apoptotic SCC25 cells was increased. Furthermore, stable silencing of PKD1 in SCC25 cells under a hypoxic condition decreased glucose uptake, lactate production and glycolytic enzyme (GLUT-1 and LDHA) expression, as well as reduced the phosphorylation of p38 MAPK. The results revealed that following inhibition of the expression of PKD1 under a hypoxic condition, the growth and metabolism of the SCC25 cells were significantly suppressed. In contrast, when PKD1 was overexpressed in SCC25 cells, the results were completely reversed, except for growth and apoptosis. Taken together, our results demonstrated that PKD1 not only regulates the hypoxic glycolytic metabolism of cancer cells via regulation of the expression of HIF-1α and glycolytic enzymes, but is also involved in the remodeling of the acidic tumor microenvironment. This study suggests that PKD1 may be a potential target for microenvironment-directed tumor biotherapy.