Leptospira spp. are fastidious and slow-growing bacteria, making recovery difficult and diagnostic sensitivity in the clinical setting low. However, collection of Leptospira isolates is valuable for epidemiological and laboratory research. Severe leptospirosis cases may present as septic shock, and the differential diagnosis often includes bacterial septicemia, leading clinicians to collect blood cultures. Here, we report the successful isolation of pathogenic Leptospira spp. from blood culture bottles (targeting aerobic bacteria incubated at 37°C) from a 64-year-old man admitted with septic shock. The patient presented with 4 days of fever, severe hypotension, transient atrial fibrillation, jaundice, and oliguric renal failure. After admission, intravenous ceftriaxone plus azithromycin was given with fluid resuscitation, norepinephrine infusion, invasive mechanical ventilation, and renal replacement therapy. He was discharged from the hospital 16 days after admission. Using the blood sample obtained on admission, the diagnosis of leptospirosis was confirmed by multiplex real-time PCR (targeting bacterial 16S rRNA and LipL32 gene). We collected 200 μL from the blood culture bottle to inoculate a 5-mL Ellinghausen, McCullough, Johnson, and Harris media supplemented with 5% fetal bovine serum. After 2 weeks of incubation at 30°C, Leptospira strains were identified and confirmed by real-time PCR. Genotyping was undertaken using the multi-locus sequence typing (MLST) scheme#1. The isolate matched with ST50 isolates in the PUbMLST database. This case provides evidence that in tropical countries, severe leptospirosis should be considered in patients who present with symptoms of sepsis. Pathogenic Leptospira may be successfully isolated from aerobic blood cultures in routine clinical settings.