Here, for the first time, we have investigated the hipBA(Xn) toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipA(Xn) toxin and HipB(Xn) antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipA(Xn) toxin, hipB(Xn) antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipA(Xn) toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipA(Xn) toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBA(Xn) TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipA(Xn) toxin, HipB(Xn) antitoxin protein, and 157 bp promoter region. Results showed that the hipBA(Xn) TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions.