Understanding the interaction between soluble factors and cells in the cellular microenvironment is critical to understanding a wide range of diseases. Microchannel culture systems provide a tool for separating diffusion and convection based transport making possible controlled studies of the effects of soluble factors in the cellular microenvironment. In this paper we compare the proliferation kinetics of cells in traditional culture flasks to those in microfluidic channels, and explore the relationship between microchannel geometry and cell proliferation. PDMS (polydimethylsiloxane) microfluidic channels were fabricated using micromolding methods. Fall armyworm ovarian cells (Sf9) were homogeneously seeded in a series of different sized microchannels and cultured under a no flow condition. The proliferation rates of Sf9 cells in all of the microchannels were slower than in the flask culture over the first 24 h of culture. The proliferation rates in the microchannels then continuously decreased reaching 5% of that in the flasks over the next 48 h and maintained this level for 5 days. This growth inhibition was reversible and influenced only by the cell seeding density and the channel height but not the channel length or width. One possible explanation for the observed dimension-dependent cell proliferation is the accumulation of different functional molecules in the diffusion dominant microchannel environment. This study provides insights into the potential effects of the diffusion of soluble factors and related effects on cell behavior in microenvironments relevant to the emerging use of microchannel culture systems.