The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from (13)C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity.