The pheromones of crane flies (Tipulidae), one of the largest families within the order Diptera (over 15,000 species), are unknown. The aim of our study was to identify the chemical compounds involved in communication in Tipula autumnalis, a representative species of the family. Female cuticular washes were found to be attractive to males in a bioassay. GC-EAD analysis revealed nine EAG-active compounds, which were identified as cuticular hydrocarbons (CHCs). Both males and females contained these CHCs, though in different ratios. The strongest antennal responses in male T. autumnalis were evoked by n-pentacosane, (Z)-9-pentacosene, and (Z, E)-6,9-pentacosadiene, which were the predominant components in females. Each of these compounds were attractive to males in the behavioral assay and are therefore attributed to the female sex pheromone of T. autumnalis. (Z)-9-tricosene and (R)-3-methylheneicosane elicited both EAG and behavioral responses in males and were abundant in washes from same-sex individuals. In addition to the compounds involved in female–male interactions, it is evident that T. autumnalis also employs CHCs in male–male interactions. The exact roles of some compounds remain undetermined. Among the semiochemicals, the established stereostructure of (Z, E)-6,9-pentacosadiene and the olfactory/behavioral effects of (R)- and (S)-3-methylheneicosane were novel findings in insects.