Nickel boride is generally used in the steel industry as a melting accelerator due to its feature of creating a protective and stable attribute at high temperatures. It is also used to improve the hardenability of the steel with boron addition in the production. Thus, safety studies and biocompatibility analysis of nickel boride should be performed comprehensively to understand the limitations of use in various areas. In the present study, nickel boride nanoparticles (Ni2B NPs) were synthesized by a single-step method and molecule characterizations were performed via the use of X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) analyses. Cytotoxicity properties of Ni2B NPs were identified on human pulmonary alveolar epithelial cells (HPAEpiC) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), and lactate dehydrogenase (LDH) assays. Illumina human ht-12 v4.0 whole-genome microarray analysis was conducted to investigate NiB(2)NPs effects on gene expression regulations of HPAEpiC cells. The database for annotation, visualization, and integrated discovery (DAVID) analysis was performed to reveal the relationship between Ni2B NP application and cellular pathway alterations. According to cytotoxicity analysis, the IC(50)value for Ni2B NP application was found as 81.99 mg/L concentration. Microarray analysis of Ni2B NP application was shown for the first time that 693 gene expression changes (FC >= 2) occurred significantly over 40.000 gene probes and Ni2B NPs were observed to affect microtubule regulation, centrosome organization, and phosphoprotein synthesis.