Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose hydrolyzing capacity is due to the absence of Asp239 and Trp47 homologues, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far the precise roles of such defective cwINVs remain unclear. In this manuscript, we report on the functional characterization of tobacco Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp239 and Trp47 homologues, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp239 and Trp47 homologues, leading to an enzyme with a distinct sucrose hydrolyzing capacity. In vitro experiments suggest that addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 (NtcwINV1) to the wall, leading to higher activities in the presence of sucrose and a more efficient interaction with its invertase inhibitor CIF. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step towards unraveling the specific roles of plant defective cwINVs.