Glucosidase II (GluII) plays a major role in regulating post-translation modification of N-linked glycoproteins. We have previously reported that the expression of glucosidase II beta subunit (GluIIβ) was significantly increased in lung tumor tissues and its suppression triggers autophagy and/or apoptosis. Here, we investigated the role of GluIIβ in cell growth, metastatic potential, and receptor tyrosine kinases (RTKs) signaling activity in lung carcinoma cell lines. CRISPR-CAS9 technology was used to knockout the GluIIβ encoding gene (PRKSH) in lung carcinoma cells. GluIIβ knockout cells exhibited drastically slower growth rates in comparison to non-target transfected cells, particularly with lower concentrations of fetal bovine serum, indicating impairment of their ability to survive under nutritional deprivation. Cell migration and anchorage-independent growth, the fundamental components of cancer cell metastasis, were significantly decreased in GluIIβ knockout cells. Knockout of GluIIβ increased the sensitivity of lung cancer cells to cisplatin but reduced their sensitivity to gefitinib. Interestingly, knocking out of GluIIβ lowered overall RTK signaling activities to less than half of those in non-target transfected cells, which could represent a novel strategy for blocking multiple RTKs in tumor cells in an effort to improve lung cancer treatment.