Halogenated boroxine is an important enzyme inhibitor with potential applications in cancer therapy. This study aimed to evaluate its effects on hematological parameters in rats in vivo and creatine kinase activity in vitro. Halogenated boroxine, administered either intraperitoneally (IP) or orally, induced route-dependent changes in hematological parameters compared with controls. IP administration significantly increased neutrophils, mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), while decreasing hematocrit (HCT). In contrast, oral administration significantly decreased lymphocytes and increased basophils, while other hematological parameters remained unchanged. These effects were more pronounced following IP administration. In vitro, halogenated boroxine exhibited competitive inhibition of creatine kinase without pre-incubation, whereas after a 30-minute pre-incubation, the inhibition pattern indicated a non-competitive mechanism. Further analysis showed a time-dependent shift in creatine kinase inhibition mechanism following pre-incubation, characterized by a decrease in maximum reaction velocity (Vmax), while the Michaelis constant (Km) remained unaltered. These findings show that halogenated boroxine modulates specific hematological parameters and inhibits creatine kinase activity, with effects depending on the route of administration and experimental conditions.