Many haloarchaea are known as polyhydroxyalkanoates (PHAs) producers, but a global and integrated view of the PHA biosynthesis is still lacking in this group of archaea. In this study, a combined proteomic and transcriptomic approach was employed in Haloarcula hispanica, a model haloarchaeon that accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under nutrient-limiting conditions with excess carbon source. First, a comprehensive proteome reference map was established for H. hispanica. A total of 936 spots representing 839 unique proteins (21.7% of the predicted proteome) were identified by MALDI-TOF/TOF PMF and MS/MS. The map was further utilized to reconstruct central metabolic pathways to facilitate functional genomic analysis in H. hispanica. The results from the proteomic and transcriptomic analysis indicated that active PHA production coordinated with the TCA cycle to maintain balanced growth in wild-type H. hispanica, which was grown in nutrient-limited medium (PHA-accumulating conditions) versus nutrient-rich medium (non-PHA-accumulating conditions). Under nutrient-limiting conditions with excess carbon source, the PHA biosynthetic genes including phaEC, phaB, and phaP were upregulated at the transcriptional level, whereas the TCA cycle and respiratory chain were downregulated. Thus, acetyl-CoA could be fed into the PHA biosynthetic pathway, leading to the accumulation of PHA granules in the cell. Simultaneously, the large amount of NADPH required during PHA accumulation was likely supplied by the C3 (pyruvate) and C4 (malate) pathway coupled with the urea cycle. When PHA biosynthesis was blocked, that is, in the PHA synthase mutant (ΔphaEC) versus wild type grown in nutrient-limited medium, the mutant might direct additional carbon and energy to the TCA cycle, but without obvious contribution to biomass accumulation. The combined approaches of proteomic and transcriptomic analysis were highly complementary, extending the physiological understanding of PHA biosynthesis and its regulation. This is the first integrated proteome and transcriptome investigation of PHA biosynthesis and regulation in haloarchaea. It has provided basic information for future systemic engineering of haloarchaea to meet industrial needs.