Monascus species have the unique ability to economically produce many secondary metabolites. However, most metabolic regulation processes in the production of secondary metabolites in Monascus remain unclear. We found that the translational inhibitor cycloheximide induced different expression patterns between the monascorubrin pigment production and the growth in Monascus pilosus. Here, we used the proteomic approach of two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight liquid chromatography-mass spectrometry (MALDI-TOF/TOF LC-MS), and tandem mass spectrometry (MS/MS) to identify the intracellular and mitochondrial proteins of M. pilosus between the cycloheximide treatment and the control. These results revealed that the cycloheximide-induced down-regulated proteins were involved in transcriptional regulation, peptide synthesis, and other metabolic processes, such as methylation of secondary metabolites. In contrast, the energy-related proteins, such as the transcriptional regulator rosAr and 1,4-alpha-glucan branching enzyme, were up-regulated as compared to the control.