This is the first integrated biological and behavioral surveillance survey among long-distance truck drivers (LDTD) in Mozambique. Using modified time-location sampling in 2012 at a key transportation junction (Inchope), we enrolled 327 male LDTD. HIV prevalence was 15.4 % (95 % confidence interval : 11.4-19.4 %, n = 318 tested). Among HIV-positive LDTD, 83.7 % did not know their status. One-third of LDTD had never tested for HIV and three-quarters had not received free condoms, lubricants or HIV literature in the past 12 months. In that same period, 61.4 % of LDTD had at least four sexual partners and 27.1 % paid for sex. Among sexually-active LDTD, 76.5 % did not use a condom at last sex. HIV was associated (p < 0.05) with primary education or lower (AOR 2.1), residence in Mozambique (AOR 2.3) and never having tested for HIV (AOR 2.2). Our findings reveal that broader coverage of HIV prevention and comprehensive care services for LDTD are urgently needed.