A novel protocol for raspberry (Rubus spp.) micropropagation in photomixotrophic conditions was optimized for the commercial genotypes Heritage, Meeker and Amity. Plant cultures were established in Temporary Immersion Bioreactors (TIBs) under a controlled environment: 550 ppmv CO2; light intensity 80 M m−2 s−1; sucrose concentrations 15 gr/L and 30 gr/L. Results showed that both CO2 flux and chlorophyll fluorescence were increased in plants cultured in TIBs with sucrose-reduced medium (15 gr/L) in comparison with those plants micropropagated in TIBs + 30 gr/L sucrose, confirming a consistent photomixotrophic stage since the 5th day of culture. Raspberry plants multiplied in TIBs + 15 gr/L sucrose demonstrated the highest values of plant size, total number of internodes, and percent of acclimatization to greenhouse conditions. Studied variables could be related to an increase of the in vitro photosynthetic activity which might prime plants for the ex vitro environment.