Abstract: Objective To investigate the thermal injury effects on human HaCaT cells under simulated microgravity environment. Methods The human HaCaT cells were collected and divided into simulated microgravity thermal injury (SMGTI) group, normal gravity thermal injury (NGTI) group, and normal gravity false injury (NGFI) group according to the random number table. Cells in NGTI and NGFI groups were cultured routinely in culture bottle, and cells in SMGTI group were cultured in the rotary cell culture system to simulate microgravity environment. Cells in SMGTI and NGTI groups were bathed in hot water of 45 ℃ for 10 minutes to make thermal injury model, and cells in NGFI group were bathed in warm water of 37 ℃ for 10 minutes to simulate thermal injury. At post injury hour (PIH) 12, cell morphology of 3 groups was observed under inverted phase contrast electron microscope. At PIH 2, 6, and 12, single cell suspension in the 3 groups was collected to detect the cell cycle by flow cytometer and the mRNA expressions of heat shock protein 70 (HSP70), matrix metalloproteinase 9 (MMP-9), and cysteine-aspartic protease 3 (caspase-3) by real time fluorescence quantitative reverse transcription polymerase chain reaction, and the experiments were repeated for 3 times. At PIH 2, 6, and 12, cell culture supernatant in the 3 groups was collected to detect the concentration of heparin-binding epidermal growth factor (HB-EGF) by enzyme linked immunosorbent assay method, the experiment was repeated for 3 times. The sample in each group and each time point was 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, least significant difference test, Kruskal-Wallis H test, and Mann-Whitney U test. Results (1) At PIH 12, cells in NGFI group showed regular shape and regular arrangement, with no cell debris. The cell shape in NGTI group was generally regular, with fewer cell debris and closer arrangement than that in NGFI group. The cells in SMGTI group showed more irregular shapes, different sizes, and dead cell debris. (2) The percentage of G1 phase cells in NGTI group was significantly higher than that in NGFI group and SMGTI group at PIH 2, respectively (P<0.05), and the percentage of G1 phase cells in NGTI group was significantly lower than that in NGFI group and SMGTI group at PIH 6 and 12, respectively (P<0.05). The percentage of G2/M phase cells in NGTI group was significantly lower than that in SMGTI group at PIH 2 (P<0.05), and the percentage of G2/M phase cells in NGTI group was significantly higher than that in NGFI group and SMGTI group at PIH 6 and 12, respectively (P<0.05). The percentage of S phase cells in NGTI group at PIH 2, 6, and 12 was significantly higher than that in SMGTI group (P<0.05), and the percentage of S phase cells in NGTI group at PIH 2 and 6 was significantly lower than that in NGFI group (P<0.05). (3) The HSP70 mRNA expressions of cells in NGTI group were 2.50±0.30 and 3.99±0.35 at PIH 2 and 6, which were significantly higher than 1.14±0.15 and 0.82±0.27 in NGFI group (P<0.05), and 1.17±0.53 and 1.65±0.59 in SMGTI group (P<0.05). The MMP-9 mRNA expression of cells in SMGTI group was significantly higher than that in NGTI group at PIH 2, 6, and 12, respectively (Z=-2.319, -2.882, -2.908, P<0.05). At each time point after injury, the mRNA expression of caspase-3 of cells in NGTI group was similar to that in NGFI group and SMGTI group, respectively (P>0.05). (4) The concentration of HB-EGF in cell culture supernatant of NGTI group was significantly lower than that in NGFI group at PIH 2, 6 and 12 (P<0.05), and the concentration of HB-EGF in cell culture supernatant of SMGTI group was significantly higher than that in NGTI group at PIH 2 and 6 (P<0.05). Conclusions The proliferation and secretion functions and expression of wound repair related protein of human HaCaT cells inflicted with thermal injury in simulated microgravity environment showed complex and diversified changes, which provide theoretical basis for further research on damage repair under weightlessness.