Maize is a major cereal crop in Tanzania and it is grown in diverse agro-ecological zones. Like other sub-Saharan countries, postharvest losses of maize during storage in Tanzania remain significantly high, especially for smallholder farmers. Unpredictable weather and poor postharvest practice contribute to rapid deterioration of grain and mold contamination, and subsequent production of mycotoxins. The purpose of this study was to assess awareness and knowledge regarding mycotoxin contamination in maize grain in three agro-ecological zones (Eastern, Central, and Northern) of Tanzania between November 2015 and February 2016. A survey using questionnaires was administered to farmers, traders, and consumers of maize. A total of 90 people (30 from each zone) were surveyed with a response rate of was 96% (87/90). In addition, several samples of maize were collected and analyzed for aflatoxin, fumonisin, and zearalenone contamination to validate the awareness and knowledge of mycotoxin contamination of maize. The result shows a high level of postharvest losses of maize mainly through insect infestation. Moreover, over 80% of the farmers, traders, and consumers of maize were unaware of mycotoxins contamination. All maize samples collected contained detected levels of mycotoxins. The maximum concentration of aflatoxins, fumonisin, and zearalenone in maize samples was 19.20 ppb,, 7.60 ppm, and 189.90 ppb respectively. Education intervention is necessary to decrease the disconnect observed between actual mycotoxin contamination and the awareness and knowledge of farmers, traders, and consumers of maize in Tanzania. Enhancing awareness and knowledge provide the opportunity to educate on post-harvest practices that reduce postharvest losses and mycotoxin of maize in Tanzania.