Methanosarcina barkeri (DSM 800) is a metabolically versatile methanogen and shows distinct metabolic status under different substrate regimes. However, the mechanisms underlying distinct transcriptional profiles under different substrate regimes remain elusive. In this study, based on transcriptional analysis, the growth performances and gene expressions of M. barkeri fed on acetate, H-2 + CO2, and methanol, respectively, were investigated. M. barkeri showed higher growth performances under methanol, followed by H-2 + CO2 and acetate, which corresponded well with the variations of gene expressions. The alpha diversity (evenness) of gene expressions was highest under the acetate regime, followed by H-2 + CO2 and methanol, and significantly and negatively correlated with growth performances. The gene co-expression analysis showed that "Energy production and conversion," "Coenzyme transport and metabolism," and "Translation, ribosomal structure, and biogenesis" showed deterministic cooperation patterns of intra- and inter-functional classes. However, "Posttranslational modification, protein turnover, chaperones" showed exclusion with other functional classes. The gene expressions and especially the relationships among them potentially drove the shifts of metabolic status under different substrate regimes. Consequently, this study revealed the diversity-related ecological strategies that a high alpha diversity probably provided more fitness and tolerance under natural environments and oppositely a low alpha diversity strengthened some specific physiological functions, as well as the co-responses of gene expressions to different substrate regimes.