Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) producing haloarchaeon, possesses four PHA synthase encoding genes,phaC,phaC1,phaC2, andphaC3. In the wild-type strain, exceptphaC, the other three genes are cryptic and not transcribed under PHA-accumulating conditions. The PhaC protein together with PhaE subunit forms the active PHA synthase and catalyzes PHBV polymerization. Previously, it was observed that the deletion of a gene namedpps-like significantly enhanced PHBV accumulation probably resulted from the upregulation ofphacluster genes (phaR-phaP-phaE-phaC). The present study demonstrated the influence ofpps-like gene deletion on the crypticphaCgenes. As revealed by qRT-PCR, the expression level of the three cryptic genes was upregulated in the Delta EPS Delta pps-like gene Delta phaCmutant. Sequential knockout of the crypticphaCgenes and fermentation experiments showed that PhaC1 followed by PhaC3 had the ability to synthesize PHBV in Delta EPS Delta pps-like gene Delta phaCmutant. Both PhaC1 and PhaC3 could complex with PhaE to form functionally active PHA synthase. However, the expression ofphaC2did not lead to PHBV synthesis. Moreover, PhaC, PhaC1, and PhaC3 exhibited distinct substrate specificity as the 3HV content in PHBV copolymers was different. The EMSA result showed that PPS-like protein might be a negative regulator ofphaC1gene by binding to its promoter region. Taken together, PhaC1 had the most pronounced effect on PHBV synthesis in Delta EPS Delta pps-like gene Delta phaCmutant and deletion ofpps-like gene released the negative effect fromphaC1expression and thereby restored PHBV accumulating ability in Delta phaCmutant.