Background: Very preterm infants (VPIs) are born with an immature gut, being sensitive to gut microbiota dysbiosis-related disease like necrotizing enterocolitis. While human milk is the best source of nutrition for VPIs, it requires fortification to meet their nutrient requirements for optimal growth. However, the optimal type of fortifier remains uncertain. Bovine colostrum (BC), rich in protein and bioactive components, may be an alternative to conventional fortifiers (CF). We aimed to investigate the distinct impacts of different bovine fortifiers, BC and CF, on the gut microbiota of VPIs. The gut microbiota of 225 VPIs who were fed human milk fortified with either BC or CF, were profiled by 16S rRNA gene amplicon sequencing of fecal samples collected before, one and two weeks of fortification. Results: Fortifier type affected the microbial community structure to a modest extent, but only explaining 1% of the variance, and no specific taxa differed between the BC and CF groups. This fortifier-derived impact was predominantly observed in VPIs born via caesarean section. Birth mode exhibited transient effects on microbial community structure shortly after birth, with caesarean section-born VPIs dominated by Firmicutes, while vaginally-born VPIs were dominated by Proteobacteria. This birth mode-derived difference diminished with age and disappeared around one month after birth. The fecal pH, increased by BC, was positively correlated with Staphylococcus and Corynebacterium, and negatively with Bifidobacterium abundance. The change in relative abundance of Staphylococcus was negatively correlated with weight gain. Conclusion: Collectively, fortification of human milk with BC or CF does influence the gut microbiota of VPIs but only to a modest extent during early life. Conversely, birth mode appears to be a significant temporary factor influencing the gut microbiota during this period. Our findings are consistent with existing literature and support the idea that the choice of fortifier has limited effects on gut microbiota development in the first month of life of VPIs.