<jats:p>The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the <jats:italic>Escherichia coli</jats:italic> production of diaponeurosporene and diapolycopene, yellow C<jats:sub>30</jats:sub> carotenoid pigments, by expressing human SQS and <jats:italic>Staphylococcus aureus</jats:italic> dehydrosqualene (C<jats:sub>30</jats:sub> carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non‐reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C<jats:sub>30</jats:sub> and C<jats:sub>40</jats:sub> carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol‐lowering drugs.