Mammalian and plant Rab1 and Rab2 are small GTPases that regulate vesicle trafficking in the endoplasmic reticulum (ER) to Golgi compartments. Little is known about their functional diversification or potential interaction. We cloned sugarcane (Saccharum officinarum L.) Rab1A and Rab2A genes and studied their functional differences by expression and complementation experiments. We found differential expression of the two genes during sugarcane leaf development: SoRab2A expression declined from the dividing base to the maturing tip of the growing leaves, whereas SoRab1A was constitutively expressed, suggesting that SoRab2A is required for cell division and expansion and SoRab1A is required for cells at all developmental stages. We used a yeast temperature sensitive ypt1-A136D mutant strain to further investigate these shared and unique functions. Ypt1 is a small GTPase that regulates vesicle transport in the same cellular location as Rab1 and Rab2. Neither SoRab1A nor SoRab2A alone could restore the growth of the mutant at restrictive temperatures when SoRab1A and SoRab2A were transformed separately. However, SoRab1A transformants maintained normal morphology and viability at non-permissive temperature, and resumed growth when returned to permissive temperature, whereas SoRab2A transformants died at non-permissive temperature, suggesting that SoRab1A function is required for a cell's viability. Mutant growth was fully restored when SoRab1A and SoRab2A were co-transformed, indicating that SoRab1A and SoRab2A complement each other and they both are needed to restore the function of ypt1-A136D. These results demonstrate that SoRab1A and SoRab2A serve distinct but overlapping functions, mostly by regulating the transportation of different sets of proteins.