AbstractOur initial goal was to evaluate the contributions of high 18:1 phosphatidylcholine and the expression level of FAE1 to the accumulation of very‐long‐chain fatty acids (VLCFAs), which have wide applications as industrial feedstocks. Unexpectedly, VLCFAs were not improved by increasing the proportions of 18:1 in fad2‐1 mutant, FAD2 artificial miRNA, and FAD2 co‐suppression lines. Expressing Arabidopsis FAE1 resulted in co‐suppression in 90% of transgenic lines, which was effectively released when it was expressed in the rdr6‐11 mutant host. When FAE1 could be highly expressed, apart from its naturally preferred product, 20:1, other saturated and polyunsaturated VLCFAs also accumulated in seeds. We postulated that overabundant FAE1 might cause the diversified VLCFA profile. When FAE1 was highly expressed, knocking down FAD2 increased the content of 20:1, suggesting that the 18:1 availability in the acyl‐CoA pool increased from the high 18:1‐PC via acyl editing. Concurrent decreases of side products like 22:1 and 20:0 in these lines suggest that increasing availability of the preferred substrate could suppress the side elongation reactions and reverse the effect of VLCFA product diversification due to overabundant FAE1. Re‐analysis of FAD2 knockdown lines indicated that increasing 18:1 led to a decrease of 22:1, which also supports the above hypothesis. These results demonstrate that 18:1 substrate could be increased by a downregulation of FAD2 and that a balance between the levels of enzyme and substrate may be crucial for engineering‐specific VLCFA products.