The diamondback moth Plutella xylostella is a major destructive pest of Brassica worldwide. P. xylostella has evolved resistance to nearly all commercial insecticides used for its control, including the most recent chemical class, diamide insecticides. Several studies show that the G4946E and I4790M mutations of ryanodine receptor (RyR) are strongly associated with diamide resistance in insects. While the pivotal functional role of G4946E in conferring diamide resistance phenotype has confirmed by several studies in different species, no direct evidence has unambiguously confirmed the functional significance of the single I4790M mutation in diamide resistance. Here, we successfully constructed a knockin homozygous strain (I4790M-KI) of P. xylostella using CRISPR/Cas9 coupled with homology directed repair approach to introduce I4790M into RyR. When compared with the background susceptible IPP-S strain, the manipulated I4790M-KI strain exhibited moderate resistance to the phthalic acid diamide flubendiamide (40.5-fold) and low resistance to anthranilic diamides chlorantraniliprole (6.0-fold) and cyantraniliprole (7.7-fold), with no changes to the toxicities of indoxacarb and β-cypermethrin. Furthermore, the acquired flubendiamide resistance was inherited in an autosomally recessive mode and significantly linked with the I4790M mutation of RyR in this I4790M-KI strain. Our findings provide in vivo functional evidence for the causality of I4790M mutation of PxRyR with moderate levels of resistance to flubendiamide in P. xylostella, and support the hypothesis that the diamide classes have different interactions with RyRs.