In order to study the synergistic mechanism of multigenes in regulation on high accumulation of carbon eighteen unsaturated fatty acids in sea buckthorn (Hippophae L.) seed oil, the line ‘Suiji 1’ with low seed oil content and the line ‘Xine 3’ with high seed oil content were selected as samples, in which these two lines had closed genetic relationship. Seeds of these two lines were harvested in six different developmental stages. Changes of the fatty acid components and relative contents in seed oils were determined by the method of gas chromatograph coupled with time of flight mass spectrometry, the expression patterns of KAR, FATB, Δ9D, KASⅡ, SAD, FAD2, FAD3, FAD7 and FAD8 genes involved in fatty acid biosynthesis were tested using qRTPCR analysis, and the effects of expression changes of multigenes on the accumulation of carbon eighteen unsaturated fatty acids were analyzed. The result showed that: (1) high accumulation of carbon eighteen unsaturated fatty acids were found in seed oils of the lines ‘Suiji 1’ and ‘Xine 3’, which were about 87.71% and 88.68% of total fatty acids, respectively. During seed development, the relative contents of oleic acid of these two lines had shown an uptrend; the relative contents of linoleic acid first decreased at initial stage of aging, then increased and finally trended nearly stable; and the relative contents of linolenic acid first increased, then decreased, and finally trended relatively stable. (2) the downregulation expression of FATB coordinated with the low expression of Δ9D genes decreased the metabolic biosynthesis of palmitic and palmitoleic acids transformed from C16∶0ACP. Relatively high expressions of KAR and KASⅡ genes improved the biosynthesis of stearic acid, which provided more precursors for the synthesis of carbon eighteen unsaturated fatty acids. (3) the continuous high expression of SAD gene catalyses stearic acid desaturation to synthesize oleic acid, and the continuous increased radio of SAD to FATB gene expressions directly improved the fatty acid desaturation rate. The expression peaks of FAD2, FAD3, FAD7 and FAD8 genes simultaneously appeared in the periods of rapid biosynthesis of linoleic and linolenic acids, and then the oleic acid was gradually desaturated to linoleic acid and linolenic acid. Our data showed that the high accumulation of carbon eighteen unsaturated fatty acids in sea buckthorn seeds are from the low expression of FATB and Δ9D genes coordinated with the high expression of KAR、KASⅡ, SAD, FAD2, FAD3, FAD7 and FAD8 genes. These results provided scientific basis for understanding the biosynthesis and accumulation mechanism of carbon eighteen unsaturated fatty acids in seeds oil, which is significant for improving ratios of different unsaturated fatty acid in plant oils.