Objective To evaluate the effect of propofol on the growth of orthotopic glioma in mice and the role of growth arrest‑specific transcript factor 5 (GAS5) in this process. Methods A total of 90 SPF‑grade healthy adult C57BL/6 mice were divided into five groups (n=18) using the random number table method: a control group (group C), a lipid emulsion treatment group (group L), a propofol treatment group (group P), a GAS5‑short hairpin RNA (shRNA) transfection followed by propofol treatment group (group GP), and a negative shRNA transfection followed by propofol treatment group (group NP). Mice in group C were intracranially transplanted with normal GL261 cells. Those in group L were intracranially transplanted with normal GL261 cells before injection of 20% lipid emulsion. Mice in group P were intracranially transplanted with normal GL261 cells before intraperitoneal injection of propofol. Groups GP and NP were intracranially transplanted with GL261 cells transfected with GAS5‑shRNA or negative shRNA before propofol intervention, respectively. During drug administration, systolic blood pressure, diastolic blood pressure, heart rate, and respiratory rate were monitored. Their survival within 40 d was recorded. After the observation period, mouse brain tissue was collected for paraffin embedding, and hematoxylin‑eosin staining was used to assess tumor formation and size of GL261 cells. Apoptosis of tumor cells was detected by terminal deoxynucleotidyl transferase‑mediated dUTP‑biotin nick end labeling (TUNEL) assay. The levels of GAS5 in tumor tissue were measured by real‑time quantitative reverse transcription polymerase chain reaction (qRT‑PCR). Ki67, c‑Myc, and glutathione‑S‑transferase Mu3 (GSTM3) positive rates in tumor tissue were determined by immunofluorescence. Results There were no statistical differences in systolic blood pressure, diastolic blood pressure, heart rate, or respiratory rate among the five groups during drug administration (all P>0.05). Tumor formation rate was 100% in all groups as confirmed by whole‑brain coronal paraffin sections. In groups C and L, the transplanted tumors were large, and crossed the midline, with numerous necrotic foci. Compared with group C, group L showed no statistical differences in 40‑day survival rate, median survival time, apoptosis indices, GAS5 levels in tumor tissue, or Ki67, c‑Myc, and GSTM3 positive rates (all P>0.05). In groups P, NP, and GP, 40‑day survival rates increased, median survival times were extended, apoptosis indices increased, GAS5 levels in tumor tissue were elevated, and Ki67, c‑Myc, and GSTM3 positive rates were reduced (all P<0.05). Compared with group P, group GP exhibited decreases in 40‑day survival rates, median survival times, apoptosis indices, GAS5 levels in tumor tissue, and increases in Ki67, c‑Myc, and GSTM3 positive rates (all P<0.05). Conclusions Propofol can inhibit the growth of orthotopic glioma in mice, and GAS5 is involved in this process.