Caerin 1.9 is a natural peptide derived from the skin secretions of the Australian tree frog (Litoria) with broad-spectrum antimicrobial and anticancer bioactivity. It improves the efficacy of a therapeutic vaccine and immune checkpoint inhibitor therapy when injected intratumorally and inhibits TC-1 tumor growth when applied topically through intact skin in a TC-1 murine tumor model. This paper investigated the pharmaceutical kinetic profile, the tissue distribution, and the acute safety investigation of Caerin 1.9 peptide in Sprague Dawley (SD) rats. The results showed that subcutaneous injection of Caerin 1.9 at 100 mg/kg is safe and does not cause mortality or organ malfunction in the recipient rats. For the consecutive injection of F3 at 10 mg/kg, the peak concentration (Cmax) of F3 displayed at 1 hr after injection in male rats was 591 ng/mL, the average drug retention time was 0.807 hr, T1/2 was 4.58 hr, and AUC0-last was 1890 h × ng/mL. In female rats, Cmax was 256 ng/mL, with an average drug retention time of 2.96 hr, T1/2 of 1.33 hr, and AUC0-last of 740 h × ng/mL. The results showed that the concentration of Caerin 1.9 in the peripheral blood peaked at 1 hour. As injected concentration increased, T1/2 extended, and Cmax, AUC0-last, and volume of distribution at a steady state all increased. After 14 days of repeated subcutaneous injection at 10.0 mg/kg, no accumulation of Caerin 1.9 in plasma was observed. The results of tissue distribution showed that the Caerin 1.9 is below the LC-MS/MS detection threshold at a minimum concentration of 40 ng/g. In conclusion, Caerin 1.9 is well tolerated in rats and could be used with current immunotherapies for better management of solid tumors and genital warts.