Five phaP family genes and one phaR gene have been identified in the genome of Burkholderia gut symbiont. PhaP proteins function as surface proteins of polyhydroxyalkanoate (PHA) granules, and PhaR protein acts as a negative regulator of PhaP biosynthesis. To address the biological roles of four phaP family genes (phaP1, phaP2, phaP3, and phaP4) and the phaR gene during insect-gut symbiont interaction, these Burkholderia mutants were fed to the second instar nymph. The ΔphaR mutant decreased the colonization ability in the host midgut compared to wild-type Burkholderia cells and negatively affected the host insect’s fitness compared with wild-type infected host. These results demonstrate that PhaR plays an important role in the biosynthesis of PHA granules and it is significantly related to the colonization of the Burkholderia gut symbiont in the host insects’ midgut