Despite their anti-inflammatory properties, role in barrier function, absorption and microbial balance in the gut, knowledge on maturational and dietary effects on intestinal short-chain fatty acids (SCFA) in neonatal piglets is scarce. Moreover, little information exists whether SCFA and lactic acid (LA) modulates gut motility at this age. The present study aimed (1) to investigate the maturational changes in the SCFA profile with and without creep feeding of piglets in the first 3 wk of life; and (2) to examine the effects of SCFA and LA on muscle contractibility in jejunal tissue from neonatal piglets ex vivo. SCFA concentrations were measured in fecal samples of 52 piglets from 10 litters collected on days 2, 6, 13, and 20 of life using gas chromatography. Half of the litters were fed a commercial creep feed from day 10 of life. The organ bath system was used to test the effect of SCFA (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate) as well as of LA and the combination of LA and SCFA on muscle contractibility in piglet's jejunum. Average daily gain of piglets was similar between groups before and after introduction of creep feed. SCFA were detectable in feces in relevant concentrations from day 2 of life and increased on day 6 in males by 3.0-fold and on day 13 in females by 1.6-fold but decreased again on day 20 in both sexes compared with day 2 (P < 0.05). Creep feeding reduced fecal SCFA by 0.6-fold on day 13 without largely modifying molar proportions, whereas it increased fecal SCFA by 0.8-fold on day 20 of life compared with the sow-reared only piglets (P < 0.05). Applying SCFA ex vivo increased the muscle contraction of the jejunum by 30% (P < 0.05). Likewise, addition of LA and the combination of LA and SCFA increased the jejunal muscle contractibility by 34.9% and 32.2%, respectively, compared with the muscle tension preaddition (P < 0.05). In conclusion, the present results for fecal SCFA in first days of life suggest high bacterial activity on milk components and emphasize the importance of SCFA for intestinal development and function. After a lag phase, creep feeding promotes fermentation in the distal colon, which may be beneficial for the gut homeostasis. Results further demonstrate the stimulating effect of SCFA and LA for jejunal motility, suggesting a role for mixing of digesta (segmentation) and digestion and absorption of nutrients as well as passage in the jejunum of neonatal piglets.