<P>To smoothen the process of <I>n</I>‐butanol formation in <I>Pseudomonas putida</I> KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance. Here, we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. <I>Pseudomonas putida</I> KT2440 was cultivated in multiple chemostat fermentations using <I>n</I>‐butanol either as sole carbon source or together with glucose. <I>Pseudomonas putida</I> KT2440 revealed maximum growth rates (μ) of 0.3？h<SUP>−1</SUP> with <I>n</I>‐butanol as sole carbon source and of 0.4？h<SUP>−1</SUP> using equal C‐molar amounts of glucose and <I>n</I>‐butanol. While C‐mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when <I>n‐</I>butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when <I>n</I>‐butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing <I>n</I>‐butanol consumption. <SUP>13</SUP>C flux analysis revealed that central metabolism was split into a glucose‐fueled Entner–Doudoroff/pentose‐phosphate pathway and an <I>n</I>‐butanol‐fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of <I>n</I>‐butanol could be unraveled, thus representing an important basis for rendering <I>P. putida</I> KT2440 from an <I>n</I>‐butanol consumer to a producer in future metabolic engineering studies.</P>