Greece experienced the largest European West Nile virus (WNV) outbreak in 2010 since the 1996 Romania epidemic. West Nile virus reemerged in southern Greece during 2017, after a 2-year hiatus of recorded human cases, and herein laboratory findings, clinical features, and geographic distribution of WNV cases are presented. Clinical specimens from patients with clinically suspected WNV infection were sent from local hospitals to the Microbiology Department of Medical School, National and Kapodistrian University of Athens, and were tested for the presence of specific anti-WNV antibodies and WNV RNA. From July to September 2017, 45 confirmed or probable WNV infection cases were identified; 43 of them with an acute/recent infection, of which 24 (55.8%) experienced WNV neuroinvasive disease (WNND). Risk factors for developing WNND included advanced age, hypertension, and diabetes mellitus. A total of four deaths (16.7%) occurred, all in elderly patients aged > 70 years. Thirty-nine cases were identified in regional units that had not been affected before (36 in Argolis and two in Corinth, northeastern Peloponnese, and one in Rethymno, Crete). The remaining four cases were reported from previously affected regional units of northwestern Peloponnese. The reemergence of WNV after a 2-year hiatus of recorded human cases and the spread of the virus in newly affected regions of the country suggests that WNV has been established in Greece and disease transmission will continue in the future. Epidemiological surveillance, intensive mosquito management programs, and public awareness campaigns about personal protective measures are crucial to the prevention of WNV transmission.