Aluminum oxide nanoparticles are listed among 14 high-priority nanomaterials published by the Organization for Economic Co-operation and Development, but limited information is available on their potential hazards. In this study, we compared the toxicity of two different aluminum oxide nanorods (AlNRs) commercially available in vivo and in vitro. Considering aspect ratio, one was 6.2 ± 0.6 (long-AlNRs) and the other was 2.1 ± 0.4 (short-AlNRs). In mice, long-AlNRs induced longer and stronger inflammatory responses than short-AlNRs, and the degree reached the maximum on day 7 for both types and decreased with time. In addition, in vitro tests were performed on six cell lines derived from potential target organs for AlNPs, HEK-293 (kidney), HACAT (skin), Chang (liver), BEAS-2B (lung), T98G (brain), and H9C2 (heart), using MTT assay, ATP assay, LDH release, and xCELLigence system. Long-AlNRs generally produced stronger toxicity than short-AlNRs, and HEK-293 cells were the most sensitive for both AlNRs, followed by BEAS-2B cells, although results from 4 kinds of toxicity tests conflicted among the cell lines. Based on these results, we suggest that toxicity of AlNRs may be related to aspect ratio (and resultant surface area). Furthermore, novel in vitro toxicity testing methods are needed to resolve questionable results caused by the unique properties of nanoparticles.