[Objective] Clostridium kluyveri genome encodes for three highly homologous thiolases. To identify the function of these three thiolases will help us to understand how Clostridium kluyveri can efficiently produce hexanoate. [Methods] The characteristics of hexanoate and butyrate production were examined via fermentation kinetics analysis. The transcriptome and reverse transcription-quantitative RCR were used to analyze the expression profiles of thiolase-encoding genes during fermentation. Thiolases from Clostridium kluyveri were heterologously expressed in Escherichia coli and their enzyme kinetic parameters were examined. [Results] Clostridium kluyveri produced butyrate, hexanoate and octanoate, of which hexanoate was the major product. Transcription analysis showed that thlA1 gene was constitutively expressed, thlA2 gene was up-regulated and thlA3 gene was down-regulated before the depletion of acetate. The three thiolase-encoding genes all had higher transcription levels, and the highest expression levels of thlA2 and thlA3 were approximately 29% and 43% that of thlA1, respectively. The enzyme kinetic parameters with four-carbon substrate demonstrated that the three thiolases from Clostridium kluyveri had similar affinity. However, the catalytic efficiency (k_cat/K_m) of ThlA1 for four-carbon substrates was lower than that of ThlA2 and ThlA3. [Conclusion] All of the three thiolases from Clostridium kluyveri had catalytic activities, and also actively expressed in vivo.