Although observations of the box jellyfish Tripedalia cystophora have been widely documented since it was first recorded in Jamaica in 1897, to date there are no published reports of its occurrence in the Gulf of Mexico. Eighteen specimens of Tripedalia cystophora (Cubozoa: Tripedaliidae), 12 and 6, respectively, were collected from a mangrove waterway near Bonita Springs, Florida, in 2010 and a brackish canal in Englewood, Florida, in 2015. Additional records from Tampa Bay (2007–2015) and Everglades City (2015), Florida, were documented with photography. Within the past few years, new records of T. cystophora from other localities, including Hawai‘i, northern Australia, and the east coast of Florida, have been reported. This study represents the first published report of the species from the Gulf of Mexico. Several other species of cubozoans have been reported from the Gulf region, but the presence of three pedalia at each corner of the swimming bell (12 tentacles total), its small size up to 13 mm, and the presence of sexually dimorphic gonads, markedly distinguishes it from other cubozoan taxa.