Nickel nanoparticles (Ni-NPs) have advantageous applications in the industry; however, little is known of their adverse effects on biological tissues. In the present study, the ground beetle was employed as a sensitive indicator for nickel oxide nanoparticles (NiO-NPs) toxicity. Adult male beetles were injected with six dose levels of NiO-NPs (0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mg/g body weight). Mortality was reported daily over 30 days under laboratory conditions to establish an LD. Nickel was detected in the testicular tissues of the beetles using X-ray analysis and transmission electronic microscopy. Beetles treated with the sublethal dose of 0.02 mg/g were selected to observe molecular, cellular, and subcellular changes. Gene transcripts of HSP70, HSP90, and MT1 were found to be increased >2.5-, 1.5-, and 2-fold, respectively, in the treated group compared with the controls. Decreased gene expression of AcPC01, AcPC02, and AcPC04 (≤1.5-, ≤2-, and < 2.5-fold, respectively, vs. controls) also were reported in the treated group. Under light microscopy, various structural changes were observed in the testicular tissues of the treated beetles. Ultrastructure observations using scanning and transmission electron microscopy showed severe damage to the subcellular organelles as well as deformities of the heads and flagella of the spermatozoa. Therefore, the present study postulated the impact of NiO-NPs in an ecological model.