The global biosphere is commonly assumed to have been less productive before the rise of complex eukaryotic ecosystems than it is today(1). However, direct evidence for this assertion is lacking. Here we present triple oxygen isotope measurements (Delta O-17) from sedimentary sulfates from the Sibley basin (Ontario, Canada) dated to about 1.4 billion years ago, which provide evidence for a less productive biosphere in the middle of the Proterozoic eon. We report what are, to our knowledge, the most-negative Delta O-17 values (down to -0.88%) observed in sulfates, except for those from the terminal Cryogenian period(2). This observation demonstrates that the mid-Proterozoic atmosphere was distinct from what persisted over approximately the past 0.5 billion years, directly reflecting a unique interplay among the atmospheric partial pressures of CO2 and O-2 and the photosynthetic O-2 flux at this time(3). Oxygenic gross primary productivity is stoichiometrically related to the photosynthetic O-2 flux to the atmosphere. Under current estimates of mid-Proterozoic atmospheric partial pressure of CO2 (2-30 times that of pre-anthropogenic levels), our modelling indicates that gross primary productivity was between about 6% and 41% of pre-anthropogenic levels if atmospheric O-2 was between 0.1-1% or 1-10% of pre-anthropogenic levels, respectively. When compared to estimates of Archaean(4-6) and Phanerozoic primary production(7), these model solutions show that an increasingly more productive biosphere accompanied the broad secular pattern of increasing atmospheric O-2 over geologic time(8).