Researches have showed that the stable carbon isotope fractionation of fatty acids in piezophilic gram-negative bacteria exhibited regular patterns with growth pressure in the deep sea, however, the connection between isotopic fractionation of fatty acids in gram-positive bacteria and pressure still kept unknown. Therefore, we studied the carbon and hydrogen isotopic fractionation of fatty acids in gram-positive bacteria Bacillus subtilis under 0.1, 10, 20, 30, 40, 50 MPa by culturing the bacterium, extracting lipids and determining two-dimensional compound specific isotope composition. The results showed that:(1) there was a good linear correlation between hydrogen isotopic fractionation of monounsaturated and branched-chain fatty acids and growth pressure (≥ 10 MPa) as the fractionation became much smaller when pressure increased;(2) carbon isotopic fractionation of branched-chain and saturated fatty acids showed good linear correlation with growth pressure (≥ 10 MPa), where the fractionation was getting much smaller as pressure increased. Additionally, δ¹³ C of monounsaturated fatty acids remained stable. The pattern of hydrogen isotopic fractionation may be attributed to the elevated reaction rate resulted from pressure which promoted the efficiency of hydrogen transformed from NADPH to fatty acids, thus, fractionation weakened. As carbon and hydrogen isotopic fractionation was pressure-dependent, therefore, caution must be taken in tracing marine organic matter sources, determining the transportation and cycles of organic matters, especially in studying hadal biogeochemistry, where stable isotopic fractionation is controlled by growth pressure and microorganisms can alter the isotope patterns of organic matters in marine sediments.